Nir to swir fluorescent compounds for imaging and detection

ABSTRACT

This disclosure provides a family of compounds that absorb and fluoresce in the short wave infrared region (SWIR, optionally 1000 nm to 1300 nm), including hydrophilic compounds that exhibit absorption and emission spectral profiles in aqueous solutions substantially similar to those observed in organic solvents such as methanol or DMSO. The compounds can be chemically linked to biomolecules including proteins, nucleic acids, and therapeutic small molecules. The compounds are useful for imaging in a variety of medical, biological and diagnostic applications, including SWIR in vivo imaging of regions of interest within a mammal.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/124,639 filed Sep. 7, 2018, which depends from and claims priority toU.S. Provisional Application No. 62/565,263 filed Sep. 29, 2017, theentire contents of each of which are incorporated herein by reference.

FIELD

This disclosure related to methods for the production of fluorescentcompounds and their use for detection or imaging in vitro, ex vivo or invivo.

BACKGROUND

Optical imaging with fluorescent molecules is a powerful imagingmodality with significant advantages over other modalities both in vitroand in vivo. Dyes that fluoresce in the far red to near-infrared (NIR)region (650-900 nm) have been widely employed for in vivo imaging due tothe superior penetration of light through tissue at these wavelengthsrelative to shorter wavelength visible or UV light which is stronglyabsorbed. NIR dyes also absorb and emit far outside of the typical rangeof tissue autofluorescence, making them extremely well suited for invitro imaging of tissues and cells. However, NIR imaging still sufferscompromised image resolution, especially at greater imaging depths, dueto the inherent light scattering properties of tissue or other turbidmedia. The magnitude of scattering by turbid media such as tissue issubstantially diminished at longer SWIR wavelengths of light, e.g.900-1700 nm which can be detected by recently available InGaAs basedcameras and detectors. More specifically, within this region there areparticular windows of excitation and emission that may result in optimalimage quality. However, the lack of availability of materials thatabsorb and/or emit light in this region and are also suitable foradaptation to in vitro or in vivo imaging applications has hobbled theadvancement of SWIR imaging of biological targets.

Known SWIR fluorescent materials include organic fluorochromes, such asIR-1048 and IR-1061, and nanomaterials such as carbon nanotubes, quantumdots and rare-earth nanocomposites. However, most known SWIR organicfluorochromes have little to no water solubility, a property necessaryfor broad in vivo use due to distribution and excretion properties, andnanoparticles generally show poor in vivo targeting qualities due tolarge size, high non-specific accumulation, poor clearance propertiesand potential toxicity. These properties also render these materialspoorly suited for translation into clinical use, where rapid clearanceis required to reduce the risk of toxicity due to long term exposure.There exists an urgent need for novel fluorescent materials with SWIRabsorbance and/or emission profiles for the development of newbiological diagnostic and clinical imaging applications. Suitablematerials with strong absorption in the SWIR region could also haveextended application into SWIR mediated photoacoustic imaging andphotodynamic therapy. Moreover, the ability to synthesize moleculeswhose optical absorbance and emission properties that align withspecific optimal windows for deep tissue or in vivo imaging would enableprecise tuning of imaging parameters with the potential for significantadvancement in the field of optical imaging.

SUMMARY

The following summary is provided to facilitate an understanding of someof the innovative features unique to the present disclosure and is notintended to be a full description. A full appreciation of the variousaspects of the disclosure can be gained by taking the entirespecification, claims, drawings, and abstract as a whole.

Compounds as provided herein may be used to form polymethine bridgedheterocycles that absorb and or emit light in the near infrared (NIR) toshortwave infrared (SWIR) regions of the electromagnetic spectrum(approximately 700 nm to approximately 2500 nm, preferably from 900 nmto 1700 nm). The compounds generally contain multiple substituents, suchas sulfonates, aryl sulfonates, C1 to C₂₄ alkyl sulfonates, taurine,carboxylates, alkylammonium, polyethylene glycol or other hydrophillicgroups that confer solubility and compatibility with biologicalenvironments or assay conditions or improve optical properties of thefluorochrome. The compounds optionally have the capability to be linked,coupled or otherwise bound to other molecules, oligonucleotides, DNAs,RNAs, PNAs, siRNAs, peptides, proteins, antibodies, nanoparticles,viruses, cells, or tissues through one or more linking or bindingsubstituents. The compounds, on their own or linked to other molecules,can be used for detection, fluorescence imaging (in vitro, ex vivo, orin vivo), photoacoustic imaging, microscopy, cytometry, immunoassays,diagnostics, photothermal therapy, or other chemical, biological, ormedical applications. The fluorescent dyes can be used in fluorescenceof imaging whole animals in NIR to SWIR wavelengths. In other aspects,the present disclosure provides methods for the production of compoundsbearing various combinations of suitable substituents. The disclosurealso provides methods and ranges for optimal imaging of tissues andwhole animals in the NIR to SWIR regions.

In certain aspects, provided are compounds having a general structure

and may include a salt of any thereof In the compounds: R₁ isindependently for each occurrence, hydrogen substituted or unsubstitutedC₁ to C₂₄ alkyl, substituted or unsubstituted alkylaryl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,sulfonate, aryl sulfonate, alkyl sulfonate, taurine, carboxylate, amine,alkylamine, arylamine, alkylammonium, arylammonium, sulfonamide,halogen, hydroxy, amide, nitro, cyano, azide, O-alkyl, S-alkyl, silyl,trialkylsilyl, 0-silyl, haloalkyl, alkylsulfhydryl, trifluoromethyl,hydrazide, substituted or unsubstituted aryl, heteroaryl, orheterocyclic (e.g. morpholine) alkynyl, carboxyalkyl, aminoalkyl,haloalkyl, azidoalkyl, amide, amino acid, or peptide or L;

L is absent or is a linker moiety, optionally bearing a functional groupor reactive group, wherein said functional group or reactive group is acarboxylate, carboxyalkyl, maleimide, succinimidyl ester, carboxamide,propargyl, azidoalkyl, alkyne, isothiocyanate, of —NH₂—OH, —SH, —SO₃H,carboxyl, —COCl, —CONHNH₂, acetoxymethyl esters, substituted andunsubstituted N-hydroxysuccinimidyl esters, substituted andunsubstituted N-hydroxysulfosuccinimido esters, nitro- or fluoro orphenol esters, azide, —COCH₂I, phosphoramidite, phthalamido, acylfluoride, acyl chloride, acyl azide, tyramide, cinnamamide,hydroxycinnamamide, aldehyde, ketone, phosphoramidite, isocyanate,isothiocyanate, sulfonyl chloride, maleimide or biotin; and

R₂ is a substituted or unsubstituted C₁ to C₂₄ alkyl, substituted orunsubstituted alkylaryl, substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl, substituted or unsubstituted alkenyl,substituted or unsubstituted alkynyl, aryl sulfonate, C₁ to C₂₄ alkylsulfonate, C₁-C₂₄ alkyl carboxylate, aryl carboxylate, C₁-C₂₄alkylamine, arylamine, C₁-C₂₄ alkylammonium, arylammonium, orsubstituted or unsubstituted polyethylene glycol.

BRIEF DESCRIPTION OF THF DRAWINGS

The aspects set forth in the drawings as provided herein areillustrative and exemplary in nature and not intended to limit thesubject matter defined by the claims. The following detailed descriptionand disclosure can be understood when read in conjunction with thefollowing drawings where like structure is indicated with like referencenumerals and in which:

FIG. 1A illustrates absorbance spectra of compound D3 according toaspects as provided herein in water or methanol;

FIG. 1B illustrates absorbance spectra of compound D19 according toaspects as provided herein in water or methanol;

FIG. 1C illustrates absorbance spectra of compound D2 according toaspects as provided herein in water or methanol;

FIG. 2 illustrates the fluorescence spectra of exemplary compound D2 invarious solvents;

FIG. 3 illustrates fluorescence spectra of various compounds accordingto aspects as provided herein relative to a reference compound IR-26;

FIG. 4 illustrates the normalized absorbance spectra of variouscompounds according to aspects as provided herein;

FIG. 5 illustrates the absorbance spectrum of an exemplaryD2-cyclo-(RGDyK) conjugate;

FIG. 6 illustrates the fluorescence spectrum of an exemplaryD2-cyclo-(RGDyK) conjugate in 1×PBS upon excitation at 980 nmillustrating an emission maximum of 1036 nm;

FIG. 7 illustrates a absorbance spectrum of compound D80 conjugated topolyethylene glycol;

FIG. 8 illustrates a fluorescence spectrum of compound D80 conjugated topolyethylene glycol;

FIG. 9 is a schematic illustration of compound D80 conjugated to anantibody;

FIG. 10 illustrates a fluorescence spectrum of exemplary compound D80conjugated to an antibody;

FIG. 11 illustrates an absorbance spectrum of exemplary compound D80conjugated atezolizumab; and

FIG. 12 illustrates a fluorescence spectrum of exemplary compound D80conjugated atezolizumab.

DETAILED DESCRIPTION

The following description of particular embodiment(s) is merelyexemplary in nature and is in no way intended to limit the scope of theinvention, its application, or uses, which may, of course, vary. Theinvention is described with relation to the non-limiting definitions andterminology included herein. These definitions and terminology are notdesigned to function as a limitation on the scope or practice of theinvention but are presented for illustrative and descriptive purposesonly. While the processes or compositions are described as an order ofindividual steps or using specific materials, it is appreciated thatsteps or materials may be interchangeable such that the description ofthe invention may include multiple parts or steps arranged in many waysas is readily appreciated by one of ordinary skill in the art.

It will be understood that, although the terms “first,” “second,”“third,” etc. may be used herein to describe various elements,components, regions, layers, and/or sections, these elements,components, regions, layers, and/or sections should not be limited bythese terms. These terms are only used to distinguish one element,component, region, layer, or section from another element, component,region, layer, or section. Thus, “a first element,” “component,”“region,” “layer,” or “section” discussed below could be termed a second(or other) element, component, region, layer, or section withoutdeparting from the teachings herein.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used herein, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms, including “at least one,” unless the content clearly indicatesotherwise. “Or” means “and/or.” As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items. It will be further understood that the terms “comprises”and/or “comprising,” or “includes” and/or “including” when used in thisspecification, specify the presence of stated features, regions,integers, steps, operations, elements, and/or components, but do notpreclude the presence or addition of one or more other features,regions, integers, steps, operations, elements, components, and/orgroups thereof. The term “or a combination thereof” means a combinationincluding at least one of the foregoing elements.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this disclosure belongs. It willbe further understood that terms such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art and thepresent disclosure, and will not be interpreted in an idealized oroverly formal sense unless expressly so defined herein.

The present disclosure provides a family of fluorochrome compounds (e.g,dyes) that absorb and/or emit light having a wavelength in the rangefrom about 700 nm to about 2500 nm, optionally in the range from about900 nm to about 1700 nm. In certain embodiments, the dyes absorb and/oremit light having a wavelength in the range from about 750 nm to about1550 nm, from about 950 nm to about 1350 nm, or from about 1000 nm toabout 1250 nm. The fluorochrome compounds or certain conjugates orderivatives thereof can, in some instances, be conjugated to othermolecules or biomolecules and are particularly useful in a variety of invitro and in vivo imaging applications.

Generally, the fluorochromes of this disclosure can be represented bythe formula P₁—B—P₂, wherein P₁ and P₂ each represent conjugatedheterocyclic or heteroaromatic moieties and B represents a bridgingfunctionality between P₁ and P₂, such as a polymethine bridge consistingof conjugated double bonded methylene chains, conjugated aryl orheteroaryl groups, thiadiazole, benzothiadiazole, bisbenzothiadiazole,or combinations thereof with suitable substituents.

In certain embodiments, the provided compounds (e.g., imaging compounds)comprises fluorescent compounds having a general structure according to(Formula I):

wherein P₁ and P₂ are heterocyclic or heteroaromatic or polycyclicmoieties such as an acridine or acridinium moiety, a pyrylium orthiopyrylium moiety, and indole, benzindole or benz[c,d]indole moiety,naphtho thiophene moiety such as a 1,8-naphtho thiophene moiety, orrelated structures and salts thereof, B is a conjugated bridging moiety,optionally a polymethine bridge including or consisting of conjugateddouble bonded methylene chains, conjugated aryl or heteroaryl groups,thiadiazole, benzothiadiazole, bisbenzothiadiazole, or other structuresas described herein and combinations thereof; R is, independently foreach occurrence, a substituent optionally as described herein; L is alinker or linking group bound to or capable of forming bonds to othermolecules, optionally drugs, peptides, proteins, antibodies, cells ortissues, or bearing a reactive group capable of forming such bonds asdescribed herein; and x and y are, for each occurrence, integers from 0to 15.

In some embodiments, the compounds provided herein may be fluorescentcompounds having a general structure according to Formula II:

wherein P₁ and P₂ are heterocyclic or heteroaromatic or polycyclicmoieties such as an acridine or acridinium moiety, a pyrylium orthiopyrylium moiety, and indole, benzindole or benz[c,d]indole moiety,naphtho thiophene moiety, or related structures and salts thereof,optionally as described herein; the conjugated bridging moiety (B inFormula I) is optionally a polymethine bridge that includes conjugateddouble bonded methylene chains, conjugated aryl or heteroaryl groups,thiadiazole, benzothiadiazole, bisbenzothiadiazole, or other structuresoptionally as described herein, and combinations thereof; Q is ahydrogen, halogen, alkyl, aryl, heteroaryl, hydroxyl, alkoxy, aryloxy,alkylthio, arylthio, nitrogen, silicon, boron, carboxy, cyano, ester,amine, amide, L or derivative thereof as described below; W is absent orrepresents a cyclic, bicyclic, polycyclic, carbocyclic, or heterocyclicgroup or derivative thereof optionally as described below; R is,independently for each occurrence, absent or a substituent optionally asdescribed herein; L is a linker or linking group bound to or capable offorming bonds to other molecules, such as drugs, peptides, proteins,antibodies, cells or tissues, or bearing a reactive group capable offorming such bonds optionally as described herein; and x, y and n are,independently for each occurrence, integers from 0 to 15.

I. Definitions

As used herein the term “biomolecule” is any molecule the generalstructure of which may be found in a living organism. The definition ofbiomolecule illustratively includes fragments of any polymeric moleculethe general structure of which may be found in a living organism.Illustrative examples of fragments include a peptide or amino acidfragment of a protein or a nucleic acid or oligonucleotide as a fragmentof a nucleic acid molecule (e.g., DNA, RNA). Illustrative examples of abiomolecule include a protein, peptide, antibody, carbohydrate, lipid,amino acid, nucleic acid, nucleotide, among others.

II. Fluorochrome Compounds

In certain embodiments, the bridge B of Formula I corresponds to one ofthe formulae shown in Table 1a below:

TABLE 1a Bridge # Structure B1

B2

B3

B4

B5

B6

B7

B8

B9

B10

B11

B12

B13

B14

B15

B16

B17

B18

B19

B20

wherein Q, X₁, X₂, R, R₁-R₄, L, W₁, and W₂ are as defined herein forformula I or II.

In certain embodiments, the bridge B of Formula I corresponds to one ofthe formulae shown in Table 1b below, or salts thereof.

TABLE 1b Bridge # Structure B30

B31

B32

B33

B34

B35

B36

B37

B38

B39

End groups (P):

In certain embodiments, P₁ and P₂ may, each independently, correspond toone of the formulae shown in Table 2a below:

TABLE 2a P # Structure P3

P4

P5

P6

P7

P8

P9

P10

P11

P12

P13

P14

P15

P16

P17

P18

P19

P20

P21

P22

P23

P24

P25

P26

Wherein X, Y, Z, R, R₁-R₁₂, Q, and W₁-W₄ are as defined herein forformula I or II.

Heteroaryl substituents X, Y and Z

A compound as provided herein optionally includes one or moreheterocycles. A heterocycle optionally includes one or more substituentsX, Y, or Z. Illustrative examples of heterocycles of P1 or P2 includingone or more substituents X, Y, or Z are illustrated but not limited tothose presented in Table 2a. In certain embodiments, X, Y, and Z are,independently, O, S, N, P, Si, C, or (C═C). It is understood that eachof X, Y, and Z, if capable, may bear additional substituents, includingbut not limited to H, C₁-24 alkyl, C₁-24 dialkyl, aryl, aryl sulfonate,C₁ to C₂₄ alkyl sulfonate, arylsulfonate, halogen, nitro, hydroxyl,N-alkyl, O-alkyl, S-alkyl, N-aryl, among others. It is furtherunderstood that substitution of X, Y, and Z may impart a net charge thatwould be paired as a salt with a suitable counterion or exist as aninner-salt (zwitterion) with another group within the same molecule.

In certain embodiments, P₁ and P₂ may, each independently, correspond toone of the formulae shown in Table 2b below, or salts thereof.

TABLE 2b P # Structure P30

P31

P32

P33

P34

P35

P36

P37

P38

P39

P40

P41

P42

P43

P44

P45

P46

P47

P48

P49

P50

P51

P52

P53

Q Substituents:

In certain embodiments of the disclosure, Q in any of the formulaeprovided herein is a hydrogen, C₁-C₂₄ alkyl, aryl, heteroaryl, polyaryl,phenyl, thienyl, furanyl, pyridyl, pyridinium, halogen, substituted orunsubstituted S-alkyl, substituted or unsubstituted S-aryl, substitutedor unsubstituted O-alkyl, substituted or unsubstituted O-aryl,substituted or unsubstituted N-alkyl, substituted or unsubstitutedN-aryl, cyano, trifluoromethyl, azidoaryl, boronic acid, boronic ester,or other. In other aspects, Q is further substituted with one or more Rgroups or L linking groups, as defined herein.

W (Rings):

In certain embodiments, W and W₁-W₄ are, independently, absent or cyclicgroups containing aliphatic or aromatic carbon, nitrogen, oxygen,sulfur, or silicon forming a 4 to 9 membered ring, optionally withfurther substituents. In other aspects, W or any one or more of W₁-W₄represent fused aromatic or heteroaromatic groups, optionally furthersubstituted with one or more R groups or L groups as defined herein. Inother aspects, W or any one or more of W₁-W₄ represent bicyclic,tricyclic or polycyclic, heterocyclic, heterobicyclic, orpolyheterocyclic moieties.

R Substituents:

In various embodiments, substituents R, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈,R₉, R₁₀, R₁₁, and R₁₂ each independently are absent or can representhydrogen, substituted or unsubstituted C₁ to C₂₄ alkyl, substituted orunsubstituted alkylaryl, substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl, substituted or unsubstituted alkenyl,substituted or unsubstituted alkynyl, sulfonate, aryl sulfonate, C₁ toC₂₄ alkyl sulfonate, taurine, carboxylate, amine, alkylamine,dialkylamine, arylamine, alkylarylamine, di(sulfoalkyl)amine,alkylammonium, arylammonium, sulfonamide, halogen, hydroxy, amide,nitro, cyano, azide, O-alkyl, S-alkyl, silyl, trialkylsilyl, O-silyl,haloalkyl, alkylsulfhydryl, trifluoromethyl, hydrazide, amino acid, orpeptide.

L Linkers:

In various aspects of the disclosure, L is absent or is a linker moietyor linking group covalently or non-covalently bound to or capable offorming covalent or non-covalent bonds to other molecules, such asdrugs, peptides, proteins, antibodies, nucleic acids, carbohydrates,lipids, biomolecules, nanoparticles, membranes, cells or tissues. Insome aspects, L may bear a reactive group capable of forming such bonds,optionally bearing a functional group such as a carboxylate,carboxyalkyl, maleimide, alkyl ester, aryl ester, succinimidyl ester,amine, carbamate, carboxamide, propargyl, azidoalkyl, isocyanate,isothiocyanate, sulfonyl chloride, pentafluorophenyl ester, acylfluoride, acyl chloride, acyl azide, tyramide, cinnamamide,hydroxycinammamide, aldehyde, ketone, phosphoramidite, phthalamido,biotin or other group that can be linked, bound or conjugated to amolecule, drug, biomolecule, peptide, protein nanoparticle, cell,tissue, etc.

In some aspects the linking, conjugation or binding of a molecule to Lis performed separately prior to use for imaging, detection, diagnostic,assay or other purposes. In other aspects, the binding or conjugation toL occurs in situ, optionally during imaging, detection, diagnostic orother assay or application, such as in a microtiter plate, in cellculture, on a microscope slide, in a cuvette, in an imager, on thesurface of a tissue, or in vivo in a living animal. In some aspects, thebinding is mediated by the action of an enzyme. In some aspects, theenzyme is horseradish peroxidase. In some aspects, the linking isirreversible. In other aspects, the linking is reversible. In otheraspects, the linking is reversible upon exposure to a reversing ordissociating reagent, or by changing temperature, pH, or ionic strength,or by exposure to light, heat, microwaves, ultrasound, metal ions,enzymes, or catalysts.

In some embodiments, L may be selected from the formulae shown in Table3, wherein E is carbon, nitrogen, sulfur, oxygen, silicon or phosphorus;F is carboxylate, carboxyalkyl, maleimide, alkyl ester, aryl ester,succinimidyl ester, amine, carbamate, carboxamide, propargyl,azidoalkyl, isocyanate, isothiocyanate, sulfonyl chloride,pentafluorophenyl ester, acyl fluoride, acyl chloride, acyl azide,tyramide, cinnamamide, hydroxycinammamide, aldehyde, ketone,phosphoramidite, phthalamido, biotin; and n is an integer between 0 and100.

TABLE 3 Linkers (L) Linker (L) Structure L1

L2

L3

L4

L5

L6

L7

L8

L9

L10

L11

L12

L13

L14

In certain aspects, the fluorochrome compounds as provided herein are asdefined in Table 4.

In certain aspects of the disclosure, P₁ and P₂ represent substitutedbenz[c,d]indole moieties. In certain aspects of the disclosure, P₁ andP₂ represent substituted benz[c,d]indole moieties optionally asillustrated in Table 4 as D1 to D20.

In certain aspects of the disclosure, P₁ and P₂ represent substitutedacridine or acridinium moieties. Optionally, P₁ and P₂ representsubstituted acridine or acridinium moieties optionally as illustrated inTable 4 as D51 to D59.

In certain aspects of the disclosure, P₁ and P₂ represent substitutedthiopyrylium or pyrylium moieties. Optionally, P₁ and P₂ representsubstituted thiopyrylium or pyrylium moieties optionally as illustratedin Table 4 as D21 to D50 and D60 to D64.

TABLE 4 Exemplary compounds according to this disclosure. D1

D2

D3

D4

D5

D6

D7

D8

D9

D10

D11

D12

D13

D14

D15

D16

D17

D18

D19

D20

D21

D22

D23

D24

D25

D26

D27

D28

D29

D30

D31

D32

D33

D34

D35

D36

D37

D38

D39

D40

D41

D42

D43

D44

D45

D46

D47

D48

D49

D50

D51

D52

D53

D54

D55

D56

D57

D58

D59

D60

D61

D62

D63

D64

D65

D66

D67

D68

D69

D70

D71

D73

D74

D75

D76

D77

D78

D79

D80

D81

D82

D85

Other exemplary dyes are shown in Table 4B:

TABLE 4B Additional exemplary compounds according to this disclosure.D72

D84

D86

TABLE 5 Exemplary compounds according to this disclosure.

No R₁ X R₂ R₃ R₄ R₅ R₆ Y R₇ Z Q T5-1 (CH₂)₁₋₁₂ H SO₃H H SO₃H H COOY(CH₂)₁₋₆H CONH-Z (CH₂)₁₋₆SO₃H H T5-2 (CH₂)₁₋₁₂ SO₃H H H H H COOY(CH₂)₁₋₆H CONH-Z (CH₂)₁₋₆SO₃H H T5-3 (CH₂)₁₋₁₂ X H SO₃H SO₃H H H COOY(CH₂)₁₋₆H CONH-Z (CH₂)₁₋₆SO₃H H T5-4 (CH₂)₁₋₁₂ X H H H SO₃H SO₃H COOY(CH₂)₁₋₆H CONH-Z (CH₂)₁₋₆SO₃H H T5-5 (CH₂)₁₋₁₂ X SO₃H H H SO₃H SO₃H COOY(CH₂)₁₋₆H CONH-Z (CH₂)₁₋₆SO₃H H T5-6 (CH₂)₁₋₁₂ X SO₃H SO₃H SO₃H H H COOY(CH₂)₁₋₆H CONH-Z (CH₂)₁₋₆SO₃H H T5-7 (CH₂)₁₋₁₂ X SO₃H SO₃H SO₃H SO₃HSO₃H COOY (CH₂)₁₋₆H CONH-Z (CH₂)₁₋₆SO₃H H T5-8 (CH₂)₁₋₁₂ X H SO₃H H SO₃HH COOY H CONH-Z (CH₂)₁₋₆SO₃H H T5-9 (CH₂)₁₋₁₂ X SO₃H H H H H COOY HCONH-Z (CH₂)₁₋₆SO₃H H T5-10 (CH₂)₁₋₁₂ X H SO₃H SO₃H H H COOY H CONH-Z(CH₂)₁₋₆SO₃H H T5-11 (CH₂)₁₋₁₂ X H H H SO₃H SO₃H COOY H CONH-Z(CH₂)₁₋₆SO₃H H T5-12 (CH₂)₁₋₁₂ X SO₃H H H SO₃H SO₃H COOY H CONH-Z(CH₂)₁₋₆SO₃H H T5-13 (CH₂)₁₋₁₂ X SO₃H SO₃H SO₃H H H COOY H CONH-Z(CH₂)₁₋₆SO₃H H T5-14 (CH₂)₁₋₁₂ X SO₃H SO₃H SO₃H SO₃H SO₃H COOY H CONH-Z(CH₂)₁₋₆SO₃H H T5-15 (CH₂)₁₋₁₂ X H SO₃H H SO₃H H COOY NSuccinimidylCONH-Z (CH₂)₁₋₆SO₃H H T5-16 (CH₂)₁₋₁₂ X SO₃H H H H H COOY NSuccinimidylCONH-Z (CH₂)₁₋₆SO₃H H T5-17 (CH₂)₁₋₁₂ X H SO₃H SO₃H H H COOYNSuccinimidyl CONH-Z (CH₂)₁₋₆SO₃H H T5-18 (CH₂)₁₋₁₂ X H H H SO₃H SO₃HCOOY NSuccinimidyl CONH-Z (CH₂)₁₋₆SO₃H H T5-19 (CH₂)₁₋₁₂ X SO₃H H H SO₃HSO₃H COOY NSuccinimidyl CONH-Z (CH₂)₁₋₆SO₃H H T5-20 (CH₂)₁₋₁₂ X SO₃HSO₃H SO₃H H H COOY NSuccinimidyl CONH-Z (CH₂)₁₋₆SO₃H H T5-21 (CH₂)₁₋₁₂ XSO₃H SO₃H SO₃H SO₃H SO₃H COOY NSuccinimidyl CONH-Z (CH₂)₁₋₆SO₃H H T5-22(CH₂)₁₋₁₂ X H SO₃H H SO₃H H CONH—Y (CH₂)₁₋₆COOH CONH-Z (CH₂)₁₋₆SO₃H HT5-23 (CH₂)₁₋₁₂ X SO₃H H H H H CONH—Y (CH₂)₁₋₆COOH CONH-Z (CH₂)₁₋₆SO₃H HT5-24 (CH₂)₁₋₁₂ X H SO₃H SO₃H H H CONH—Y (CH₂)₁₋₆COOH CONH-Z(CH₂)₁₋₆SO₃H H T5-25 (CH₂)₁₋₁₂ X H H H SO₃H SO₃H CONH—Y (CH₂)₁₋₆COOHCONH-Z (CH₂)₁₋₆SO₃H H T5-26 (CH₂)₁₋₁₂ X SO₃H H H SO₃H SO₃H CONH—Y(CH₂)₁₋₆COOH CONH-Z (CH₂)₁₋₆SO₃H H T5-27 (CH₂)₁₋₁₂ X SO₃H SO₃H SO₃H H HCONH—Y (CH₂)₁₋₆COOH CONH-Z (CH₂)₁₋₆SO₃H H T5-28 (CH₂)₁₋₁₂ X SO₃H SO₃HSO₃H SO₃H SO₃H CONH—Y (CH₂)₁₋₆COOH CONH-Z (CH₂)₁₋₆SO₃H H T5-29 (CH₂)₁₋₁₂X H SO₃H H SO₃H H CONH—Y (CH₂)₁₋₆COO—SU CONH-Z (CH₂)₁₋₆SO₃H H T5-30(CH₂)₁₋₁₂ X SO₃H H H H H CONH—Y (CH₂)₁₋₆COO—SU CONH-Z (CH₂)₁₋₆SO₃H HT5-31 (CH₂)₁₋₁₂ X H SO₃H SO₃H H H CONH—Y (CH₂)₁₋₆COO—SU CONH-Z(CH₂)₁₋₆SO₃H H T5-32 (CH₂)₁₋₁₂ X H H H SO₃H SO₃H CONH—Y (CH₂)₁₋₆COO—SUCONH-Z (CH₂)₁₋₆SO₃H H T5-33 (CH₂)₁₋₁₂ X SO₃H H H SO₃H SO₃H CONH—Y(CH₂)₁₋₆COO—SU CONH-Z (CH₂)₁₋₆SO₃H H T5-34 (CH₂)₁₋₁₂ X SO₃H SO₃H SO₃H HH CONH—Y (CH₂)₁₋₆COO—SU CONH-Z (CH₂)₁₋₆SO₃H H T5-35 (CH₂)₁₋₁₂ X SO₃HSO₃H SO₃H SO₃H SO₃H CONH—Y (CH₂)₁₋₆COO—SU CONH-Z (CH₂)₁₋₆SO₃H H T5-36(CH₂)₁₋₁₂ X H SO₃H H SO₃H H CONH—Y (CH₂)₁₋₆CO—Mal CONH-Z (CH₂)₁₋₆SO₃H HT5-37 (CH₂)₁₋₁₂ X SO₃H H H H H CONH—Y (CH₂)₁₋₆CO—Mal CONH-Z (CH₂)₁₋₆SO₃HH T5-38 (CH₂)₁₋₁₂ X H SO₃H SO₃H H H CONH—Y (CH₂)₁₋₆CO—Mal CONH-Z(CH₂)₁₋₆SO₃H H T5-39 (CH₂)₁₋₁₂ X H H H SO₃H SO₃H CONH—Y (CH₂)₁₋₆CO—MalCONH-Z (CH₂)₁₋₆SO₃H H T5-40 (CH₂)₁₋₁₂ X SO₃H H H SO₃H SO₃H CONH—Y(CH₂)₁₋₆CO—Mal CONH-Z (CH₂)₁₋₆SO₃H H T5-41 (CH₂)₁₋₁₂ X SO₃H SO₃H SO₃H HH CONH—Y (CH₂)₁₋₆CO—Mal CONH-Z (CH₂)₁₋₆SO₃H H T5-42 (CH₂)₁₋₁₂ X SO₃HSO₃H SO₃H SO₃H SO₃H CONH—Y (CH₂)₁₋₆CO—Mal CONH-Z (CH₂)₁₋₆SO₃H H T5-43(CH₂)₁₋₆ Ph-SO₃H SO₃H H SO₃H H CONH—Y (CH₂)₁₋₆COOH CONH-Z (CH₂)₁₋₆SO₃H HT5-44 (CH₂)₁₋₆ Ph-SO₃H H H H H CONH—Y (CH₂)₁₋₆COOH CONH-Z (CH₂)₁₋₆SO₃H HT5-45 (CH₂)₁₋₆ Ph-SO₃H SO₃H SO₃H H H CONH—Y (CH₂)₁₋₆COOH CONH-Z(CH₂)₁₋₆SO₃H H T5-46 (CH₂)₁₋₆ Ph-SO₃H H H SO₃H SO₃H CONH—Y (CH₂)₁₋₆COOHCONH-Z (CH₂)₁₋₆SO₃H H T5-47 (CH₂)₁₋₆ Ph-SO₃H H H SO₃H SO₃H CONH—Y(CH₂)₁₋₆COOH CONH-Z (CH₂)₁₋₆SO₃H H T5-48 (CH₂)₁₋₆ Ph-SO₃H SO₃H SO₃H H HCONH—Y (CH₂)₁₋₆COOH CONH-Z (CH₂)₁₋₆SO₃H H T5-49 (CH₂)₁₋₆ Ph-SO₃H SO₃HSO₃H SO₃H SO₃H CONH—Y (CH₂)₁₋₆COOH CONH-Z (CH₂)₁₋₆SO₃H H T5-50 (CH₂)₁₋₆Ph-SO₃H SO₃H H SO₃H H CONH—Y (CH₂)₁₋₆CO—SU CONH-Z (CH₂)₁₋₆SO₃H H T5-51(CH₂)₁₋₆ Ph-SO₃H H H H H CONH—Y (CH₂)₁₋₆CO—SU CONH-Z (CH₂)₁₋₆SO₃H HT5-52 (CH₂)₁₋₆ Ph-SO₃H SO₃H SO₃H H H CONH—Y (CH₂)₁₋₆CO—SU CONH-Z(CH₂)₁₋₆SO₃H H T5-53 (CH₂)₁₋₆ Ph-SO₃H H H SO₃H SO₃H CONH—Y (CH₂)₁₋₆CO—SUCONH-Z (CH₂)₁₋₆SO₃H H T5-54 (CH₂)₁₋₆ Ph-SO₃H H H SO₃H SO₃H CONH—Y(CH₂)₁₋₆CO—SU CONH-Z (CH₂)₁₋₆SO₃H H T5-55 (CH₂)₁₋₆ Ph-SO₃H SO₃H SO₃H H HCONH—Y (CH₂)₁₋₆CO—SU CONH-Z (CH₂)₁₋₆SO₃H H T5-56 (CH₂)₁₋₆ Ph-SO₃H SO₃HSO₃H SO₃H SO₃H CONH—Y (CH₂)₁₋₆CO—SU CONH-Z (CH₂)₁₋₆SO₃H H T5-57 (CH₂)₁₋₆Ph-SO₃H SO₃H H SO₃H H CONH—Y (CH₂)₁₋₆CO—Mal CONH-Z (CH₂)₁₋₆SO₃H H T5-58(CH₂)₁₋₆ Ph-SO₃H H H H H CONH—Y (CH₂)₁₋₆CO—Mal CONH-Z (CH₂)₁₋₆SO₃H HT5-59 (CH₂)₁₋₆ Ph-SO₃H SO₃H SO₃H H H CONH—Y (CH₂)₁₋₆CO—Mal CONH-Z(CH₂)₁₋₆SO₃H H T5-60 (CH₂)₁₋₆ Ph-SO₃H H H SO₃H SO₃H CONH—Y(CH₂)₁₋₆CO—Mal CONH-Z (CH₂)₁₋₆SO₃H H T5-61 (CH₂)₁₋₆ Ph-SO₃H H H SO₃HSO₃H CONH—Y (CH₂)₁₋₆CO—Mal CONH-Z (CH₂)₁₋₆SO₃H H T5-62 (CH₂)₁₋₆ Ph-SO₃HSO₃H SO₃H H H CONH—Y (CH₂)₁₋₆CO—Mal CONH-Z (CH₂)₁₋₆SO₃H H T5-63 (CH₂)₁₋₆Ph-SO₃H SO₃H SO₃H SO₃H SO₃H CONH—Y (CH₂)₁₋₆CO—Mal CONH-Z (CH₂)₁₋₆SO₃H HOSu = N-Hydroxysuccinimidyl; MAL = N-(2-(N-maleimido)-ethyl)-amide

TABLE 6 Exemplary compounds according to this disclosure.

R1a = R1b = No (CH₂)₁₋₁₂- (CH₂)₁₋₁₂- R₂ R₃ R₄ R₅ R₆ Y R₇ Z Q T6-1 COOHSO₃H SO₃H H SO₃H H H H — H T6-2 COOH SO₃H H H H H H H H T6-3 COOH SO₃HSO₃H SO₃H H H H H H T6-4 COOH H H H SO₃H SO₃H H H H T6-5 COOH SO₃H H HSO₃H SO₃H H H H T6-6 COOH H SO₃H SO₃H H H H H H T6-7 COOH SO₃H SO₃H SO₃HSO₃H SO₃H H H H T6-8 COOH SO₃H SO₃H H SO₃H H H H — Cl T6-9 COOH SO₃H H HH H H H Cl T6-10 COOH SO₃H SO₃H SO₃H H H H H CI T6-11 COOH H H H SO₃HSO₃H H H Cl T6-12 COOH SO₃H H H SO₃H SO₃H H H Cl T6-13 COOH H SO₃H SO₃HH H H H Cl T6-14 COOH SO₃H SO₃H SO₃H SO₃H SO₃H H H Cl T6-15 COOH SO₃HSO₃H H SO₃H H H H — S(CH₂)₁₋₆SO₃H T6-16 COOH SO₃H H H H H H HS(CH₂)₁₋₆SO₃H T6-17 COOH SO₃H SO₃H SO₃H H H H H S(CH₂)₁₋₆SO₃H T6-18 COOHH H H SO₃H SO₃H H H S(CH₂)₁₋₆SO₃H T6-19 COOH SO₃H H H SO₃H SO₃H H HS(CH₂)₁₋₆SO₃H T6-20 COOH H SO₃H SO₃H H H H H S(CH₂)₁₋₆SO₃H T6-21 COOHSO₃H SO₃H SO₃H SO₃H SO₃H H H S(CH₂)₁₋₆SO₃H T6-22 CO—OSU SO₃H SO₃H H SO₃HH H H — S(CH₂)₁₋₆SO₃H T6-23 CO—OSU SO₃H H H H H H H S(CH₂)₁₋₆SO₃H T6-24CO—OSU SO₃H SO₃H SO₃H H H H H S(CH₂)₁₋₆SO₃H T6-25 CO—OSU H H H SO₃H SO₃HH H S(CH₂)₁₋₆SO₃H T6-26 CO—OSU SO₃H H H SO₃H SO₃H H H S(CH₂)₁₋₆SO₃HT6-27 CO—OSU H SO₃H SO₃H H H H H S(CH₂)₁₋₆SO₃H T6-28 CO—OSU SO₃H SO₃HSO₃H SO₃H SO₃H H H S(CH₂)₁₋₆SO₃H T6-29 COOH SO₃H SO₃H H SO₃H H H H —S-Ph-SO₃H T6-30 COOH SO₃H H H H H H H S-Ph-SO₃H T6-31 COOH SO₃H SO₃HSO₃H H H H H S-Ph-SO₃H T6-32 COOH H H H SO₃H SO₃H H H S-Ph-SO₃H T6-33COOH SO₃H H H SO₃H SO₃H H H S-Ph-SO₃H T6-34 COOH H SO₃H SO₃H H H H HS-Ph-SO₃H T6-35 COOH SO₃H SO₃H SO₃H SO₃H SO₃H H H S-Ph-SO₃H T6-36 CO—OSUSO₃H SO₃H H SO₃H H H H — S-Ph-SO₃H T6-37 CO—OSU SO₃H H H H H H HS-Ph-SO₃H T6-38 CO—OSU SO₃H SO₃H SO₃H H H H H S-Ph-SO₃H T6-39 CO—OSU H HH SO₃H SO₃H H H S-Ph-SO₃H T6-40 CO—OSU SO₃H H H SO₃H SO₃H H H S-Ph-SO₃HT6-41 CO—OSU H SO₃H SO₃H H H H H S-Ph-SO₃H T6-42 CO—OSU SO₃H SO₃H SO₃HSO₃H SO₃H H H S-Ph-SO₃H T6-43 CO—MAL SO₃H SO₃H H SO₃H H H H — S-Ph-SO₃HT6-44 CO—MAL SO₃H H H H H H H S-Ph-SO₃H T6-45 CO—MAL SO₃H SO₃H SO₃H H HH H S-Ph-SO₃H T6-46 CO—MAL H H H SO₃H SO₃H H H S-Ph-SO₃H T6-47 CO—MALSO₃H H H SO₃H SO3H H H S-Ph-SO₃H T6-48 CO—MAL H SO₃H SO₃H H H H HS-Ph-SO₃H T6-49 CO—MAL SO₃H SO₃H SO₃H SO₃H SO₃H H H S-Ph-SO₃H T6-50 COOHSO₃H SO₃H H SO₃H H H H — S(CH₂)₁₋₆SO₃H T6-51 COOH SO₃H H H H H H HS(CH₂)₁₋₆SO₃H T6-52 COOH SO₃H SO₃H SO₃H H H H H S(CH₂)₁₋₆SO₃H T6-53 COOHH H H SO₃H SO₃H H H S(CH₂)₁₋₆SO₃H T6-54 COOH SO₃H H H SO₃H SO₃H H HS(CH₂)₁₋₆SO₃H T6-55 COOH H SO₃H SO₃H H H H H S(CH₂)₁₋₆SO₃H T6-56 COOHSO₃H SO₃H SO₃H SO₃H SO₃H H H S(CH₂)₁₋₆SO₃H T6-57 CO—OSU SO₃H SO₃H H SO₃HH H H — S(CH₂)₁₋₆SO₃H T6-58 CO—OSU SO₃H H H H H H H S(CH₂)₁₋₆SO₃H T6-59CO—OSU SO₃H SO₃H SO₃H H H H H S(CH₂)₁₋₆SO₃H T6-60 CO—OSU H H H SO₃H SO₃HH H S(CH₂)₁₋₆SO₃H T6-61 CO—OSU SO₃H H H SO₃H SO₃H H H S(CH₂)₁₋₆SO₃HT6-62 CO—OSU H SO₃H SO₃H H H H H S(CH₂)₁₋₆SO₃H T6-63 CO—OSU SO₃H SO₃HSO₃H SO₃H SO₃H H H S(CH₂)₁₋₆SO₃H T6-64 COOH SO₃H SO₃H H SO₃H H H H —O-Ph-SO₃H T6-65 COOH SO₃H H H H H H H O-Ph-SO₃H T6-66 COOH SO₃H SO₃HSO₃H H H H H O-Ph-SO₃H T6-67 COOH H H H SO₃H SO₃H H H O-Ph-SO₃H T6-68COOH SO₃H H H SO₃H SO₃H H H O-Ph-SO₃H T6-69 COOH H SO₃H SO₃H H H H HO-Ph-SO₃H T6-70 COOH SO₃H SO₃H SO₃H SO₃H SO₃H H H O-Ph-SO₃H T6-71 CO—OSUSO₃H SO₃H H SO₃H H H H — O-Ph-SO₃H T6-72 CO—OSU SO₃H H H H H H HO-Ph-SO₃H T6-73 CO—OSU SO₃H SO₃H SO₃H H H H H O-Ph-SO₃H T6-74 CO—OSU H HH SO₃H SO₃H H H O-Ph-SO₃H T6-75 CO—OSU SO₃H H H SO₃H SO₃H H H O-Ph-SO₃HT6-76 CO—OSU H SO₃H SO₃H H H H H O-Ph-SO₃H T6-77 CO—OSU SO₃H SO₃H SO₃HSO₃H SO₃H H H O-Ph-SO₃H T6-78 CO—MAL SO₃H SO₃H H SO₃H H H H — O-Ph-SO₃HT6-79 CO—MAL SO₃H H H H H H H O-Ph-SO₃H T6-80 CO—MAL SO₃H SO₃H SO₃H H HH H O-Ph-SO₃H T6-81 CO—MAL H H H SO₃H SO₃H H H O-Ph-SO₃H T6-82 CO—MALSO₃H H H SO₃H SO₃H H H O-Ph-SO₃H T6-83 CO—MAL H SO₃H SO₃H H H H HO-Ph-SO₃H T6-84 CO—MAL SO₃H SO₃H SO₃H SO₃H SO₃H H H O-Ph-SO₃H T6-85 SO₃HSO₃H SO₃H H SO₃H H H H — Cl T6-86 SO₃H SO₃H H H H H H H Cl T6-87 SO₃HSO₃H SO₃H SO₃H H H H H Cl T6-88 SO₃H SO₃H H H SO₃H SO₃H H H Cl T6-89SO₃H SO₃H H H SO₃H SO₃H H H Cl T6-90 SO₃H SO₃H SO₃H SO₃H H H H H ClT6-91 SO₃H SO₃H SO₃H SO₃H SO₃H SO₃H H H Cl T6-92 SO₃H SO₃H SO₃H H SO₃H HH H — S(CH₂)₁₋₆COOH T6-93 SO₃H SO₃H H H H H H H S(CH₂)₁₋₆COOH T6-94 SO₃HSO₃H SO₃H SO₃H H H H H S(CH₂)₁₋₆COOH T6-95 SO₃H SO₃H H H SO₃H SO₃H H HS(CH₂)₁₋₆COOH T6-96 SO₃H SO₃H H H SO₃H SO₃H H H S(CH₂)₁₋₆COOH T6-97 SO₃HSO₃H SO₃H SO₃H H H H H S(CH₂)₁₋₆COOH T6-98 SO₃H SO₃H SO₃H SO₃H SO₃H SO₃HH H S(CH₂)₁₋₆COOH T6-99 SO₃H SO₃H SO₃H H SO₃H H H H — S(CH₂)₁₋₆CO—OSUT6-100 SO₃H SO₃H H H H H H H S(CH₂)₁₋₆CO—OSU T6-101 SO₃H SO₃H SO₃H SO₃HH H H H S(CH₂)₁₋₆CO—OSU T6-102 SO₃H SO₃H H H SO₃H SO₃H H HS(CH₂)₁₋₆CO—OSU T6-103 SO₃H SO₃H H H SO₃H SO₃H H H S(CH₂)₁₋₆CO—OSUT6-104 SO₃H SO₃H SO₃H SO₃H H H H H S(CH₂)₁₋₆CO—OSU T6-105 SO₃H SO₃H SO₃HSO₃H SO₃H SO₃H H H S(CH₂)₁₋₆CO—OSU T6-106 SO₃H SO₃H SO₃H H SO₃H H H H —S(CH₂)₁₋₆CO—MAL T6-107 SO₃H SO₃H H H H H H H S(CH₂)₁₋₆CO—MAL T6-108 SO₃HSO₃H SO₃H SO₃H H H H H S(CH₂)₁₋₆CO—MAL T6-109 SO₃H SO₃H H H SO₃H SO₃H HH S(CH₂)₁₋₆CO—MAL T6-110 SO₃H SO₃H H H SO₃H SO₃H H H S(CH₂)₁₋₆CO—MALT6-111 SO₃H SO₃H SO₃H SO₃H H H H H S(CH₂)₁₋₆CO—MAL T6-112 SO₃H SO₃H SO₃HSO₃H SO₃H SO₃H H H S(CH₂)₁₋₆CO—MAL T6-113 SO₃H SO₃H SO₃H H SO₃H H H H —S-Ph-(CH₂)₀₋₆COOH T6-114 SO₃H SO₃H H H H H H H S-Ph-(CH₂)₀₋₆COOH T6-115SO₃H SO₃H SO₃H SO₃H H H H H S-Ph-(CH₂)₀₋₆COOH T6-116 SO₃H SO₃H H H SO₃HSO₃H H H S-Ph-(CH₂)₀₋₆COOH T6-117 SO₃H SO₃H H H SO₃H SO₃H H HS-Ph-(CH₂)₀₋₆COOH T6-118 SO₃H SO₃H SO₃H SO₃H H H H H S-Ph-(CH₂)₀₋₆COOHT6-119 SO₃H SO₃H SO₃H SO₃H SO₃H SO₃H H H S-Ph-(CH₂)₀₋₆COOH T6-120 SO₃HSO₃H SO₃H H SO₃H H H H — S-Ph-(CH₂)₀₋₆CO—OSU T6-121 SO₃H SO₃H H H H H HH S-Ph-(CH₂)₀₋₆CO—OSU T6-122 SO₃H SO₃H SO₃H SO₃H H H H HS-Ph-(CH₂)₀₋₆CO—OSU T6-123 SO₃H SO₃H H H SO₃H SO₃H H HS-Ph-(CH₂)₀₋₆CO—OSU T6-124 SO₃H SO₃H H H SO₃H SO₃H H HS-Ph-(CH₂)₀₋₆CO—OSU T6-125 SO₃H SO₃H SO₃H SO₃H H H H HS-Ph-(CH₂)₀₋₆CO—OSU T6-126 SO₃H SO₃H SO₃H SO₃H SO₃H SO₃H H HS-Ph-(CH₂)₀₋₆CO—OSU T6-127 SO₃H SO₃H SO₃H H SO₃H H H H —S-Ph-(CH₂)₀₋₆CO—MAL T6-128 SO₃H SO₃H H H H H H H S-Ph-(CH₂)₀₋₆CO—MALT6-129 SO₃H SO₃H SO₃H SO₃H H H H H S-Ph-(CH₂)₀₋₆CO—MAL T6-130 SO₃H SO₃HH H SO₃H SO₃H H H S-Ph-(CH₂)₀₋₆CO—MAL T6-131 SO₃H SO₃H H H SO₃H SO₃H H HS-Ph-(CH₂)₀₋₆CO—MAL T6-132 SO₃H SO₃H SO₃H SO₃H H H H HS-Ph-(CH₂)₀₋₆CO—MAL T6-133 SO₃H SO₃H SO₃H SO₃H SO₃H SO₃H H HS-Ph-(CH₂)₀₋₆CO—MAL T6-134 SO₃H SO₃H SO₃H H SO₃H H H H —O-Ph-(CH₂)₀₋₆COOH T6-135 SO₃H SO₃H H H H H H H O-Ph-(CH₂)₀₋₆COOH T6-136SO₃H SO₃H SO₃H SO₃H H H H H O-Ph-(CH₂)₀₋₆COOH T6-137 SO₃H SO₃H H H SO₃HSO₃H H H O-Ph-(CH₂)₀₋₆COOH T6-138 SO₃H SO₃H H H SO₃H SO₃H H HO-Ph-(CH₂)₀₋₆COOH T6-139 SO₃H SO₃H SO₃H SO₃H H H H H O-Ph-(CH₂)₀₋₆COOHT6-140 SO₃H SO₃H SO₃H SO₃H SO₃H SO₃H H H O-Ph-(CH₂)₀₋₆COOH T6-141 SO₃HSO₃H SO₃H H SO₃H H H H — O-Ph-(CH₂)₀₋₆CO—OSU T6-142 SO₃H SO₃H H H H H HH O-Ph-(CH₂)₀₋₆CO—OSU T6-143 SO₃H SO₃H SO₃H SO₃H H H H HO-Ph-(CH₂)₀₋₆CO—OSU T6-144 SO₃H SO₃H H H SO₃H SO₃H H HO-Ph-(CH₂)₀₋₆CO—OSU T6-145 SO₃H SO₃H H H SO₃H SO₃H H HO-Ph-(CH₂)₀₋₆CO—OSU T6-146 SO₃H SO₃H SO₃H SO₃H H H H HO-Ph-(CH₂)₀₋₆CO—OSU T6-147 SO₃H SO₃H SO₃H SO₃H SO₃H SO₃H H HO-Ph-(CH₂)₀₋₆CO—OSU T6-148 SO₃H SO₃H SO₃H H SO₃H H H H —O-Ph-(CH₂)₀₋₆CO—MAL T6-149 SO₃H SO₃H H H H H H H O-Ph-(CH₂)₀₋₆CO—MALT6-150 SO₃H SO₃H SO₃H SO₃H H H H H O-Ph-(CH₂)₀₋₆CO—MAL T6-151 SO₃H SO₃HH H SO₃H SO₃H H H O-Ph-(CH₂)₀₋₆CO—MAL T6-152 SO₃H SO₃H H H SO₃H SO₃H H HO-Ph-(CH₂)₀₋₆CO—MAL T6-153 SO₃H SO₃H SO₃H SO₃H H H H HO-Ph-(CH₂)₀₋₆CO—MAL T6-154 SO₃H SO₃H SO₃H SO₃H SO₃H SO₃H H HO-Ph-(CH₂)₀₋₆CO—MAL OSu = N-Hydroxysuccinimidyl; MAL =N-(2-(N-maleimido)-ethyl)-amide

TABLE 7 Exemplary compounds according to this disclosure.

R_(1a) = R_(1b) = No (CH₂)₁₋₁₂- (CH₂)₁₋₁₂- R₂ R₃ R₄ R₅ T7-1 COOH SO₃HSO₃H H SO₃H H T7-2 COOH SO₃H H H H H T7-3 COOH SO₃H SO₃H SO₃H H H T7-4COOH H H H SO₃H SO₃H T7-5 COOH SO₃H H H SO₃H SO₃H T7-6 COOH H SO₃H SO₃HH H T7-7 COOH SO₃H SO₃H SO₃H SO₃H SO₃H T7-8 CO—OSU SO₃H SO₃H H SO₃H HT7-9 CO—OSU SO₃H H H H H T7-10 CO—OSU SO₃H SO₃H SO₃H H H T7-11 CO—OSU HH H SO₃H SO₃H T7-12 CO—OSU SO₃H H H SO₃H SO₃H T7-13 CO—OSU H SO₃H SO₃H HH T7-14 CO—OSU SO₃H SO₃H SO₃H SO₃H SO₃H T7-15 CO—MAL SO₃H SO₃H H SO₃H HT7-16 CO—MAL SO₃H H H H H T7-17 CO—MAL SO₃H SO₃H SO₃H H H T7-18 CO—MAL HH H SO₃H SO₃H T7-19 CO—MAL SO₃H H H SO₃H SO₃H T7-20 CO—MAL H SO₃H SO₃H HH T7-21 CO—MAL SO₃H SO₃H SO₃H SO₃H SO₃H OSu = N-Hydroxysuccinimidyl; MAL= N-(2-(N-maleimido)-ethyl)-amide

TABLE 8 Exemplary compounds according to this disclosure.Napthothiophene based SWIR Dyes

No R₂ R₃ R₄ R₅ R₆ Y R₇ Z Q T8-1 SO₃H H SO₃H H COOY (CH₂)₁₋₆H CONH-Z(CH₂)₁₋₆SO₃H H T8-2 H H H H COOY (CH₂)₁₋₆H CONH-Z (CH₂)₁₋₆SO₃H H T8-3SO₃H SO₃H H H COOY (CH₂)₁₋₆H CONH-Z (CH₂)₁₋₆SO₃H H T8-4 H H SO₃H SO₃HCOOY (CH₂)₁₋₆H CONH-Z (CH₂)₁₋₆SO₃H H T8-5 SO₃H SO₃H SO₃H SO₃H COOY(CH₂)₁₋₆H CONH-Z (CH₂)₁₋₆SO₃H H T8-6 SO₃H H SO₃H H COOY H CONH-Z(CH₂)₁₋₆SO₃H H T8-7 H H H H COOY H CONH-Z (CH₂)₁₋₆SO₃H H T8-8 SO₃H SO₃HH H COOY H CONH-Z (CH₂)₁₋₆SO₃H H T8-9 H H SO₃H SO₃H COOY H CONH-Z(CH₂)₁₋₆SO₃H H T8-10 SO₃H SO₃H SO₃H SO₃H COOY H CONH-Z (CH₂)₁₋₆SO₃H HT8-11 SO₃H H SO₃H H COOY N-Succinimidyl CONH-Z (CH₂)₁₋₆SO₃H H T8-12 H HH H COOY N-Succinimidyl CONH-Z (CH₂)₁₋₆SO₃H H T8-13 SO₃H SO₃H H H COOYN-Succinimidyl CONH-Z (CH₂)₁₋₆SO₃H H T8-14 H H SO₃H SO₃H COOYN-Succinimidyl CONH-Z (CH₂)₁₋₆SO₃H H T8-15 SO₃H SO₃H SO₃H SO₃H COOYN-Succinimidyl CONH-Z (CH₂)₁₋₆SO₃H H T8-16 SO₃H H SO₃H H CONH—Y(CH₂)₁₋₆COOH CONH-Z (CH₂)₁₋₆SO₃H H T8-17 H H H H CONH—Y (CH₂)₁₋₆COOHCONH-Z (CH₂)₁₋₆SO₃H H T8-18 SO₃H SO₃H H H CONH—Y (CH₂)₁₋₆COOH CONH-Z(CH₂)₁₋₆SO₃H H T8-19 H H SO₃H SO₃H CONH—Y (CH₂)₁₋₆COOH CONH-Z(CH₂)₁₋₆SO₃H H T8-20 SO₃H SO₃H SO₃H SO₃H CONH—Y (CH₂)₁₋₆COOH CONH-Z(CH₂)₁₋₆SO₃H H T8-21 SO₃H H SO₃H H CONH—Y (CH₂)₁₋₆CO—OSu CONH-Z(CH₂)₁₋₆SO₃H H T8-22 H H H H CONH—Y (CH₂)₁₋₆CO—OSu CONH-Z (CH₂)₁₋₆SO₃H HT8-23 SO₃H SO₃H H H CONH—Y (CH₂)₁₋₆CO—OSu CONH-Z (CH₂)₁₋₆SO₃H H T8-24 HH SO₃H SO₃H CONH—Y (CH₂)₁₋₆CO—OSu CONH-Z (CH₂)₁₋₆SO₃H H T8-25 SO₃H SO₃HSO₃H SO₃H CONH—Y (CH₂)₁₋₆CO—OSu CONH-Z (CH₂)₁₋₆SO₃H H T8-26 SO₃H H SO₃HH CONH—Y (CH₂)₁₋₆CO—Mal CONH-Z (CH₂)₁₋₆SO₃H H T8-27 H H H H CONH—Y(CH₂)₁₋₆CO—Mal CONH-Z (CH₂)₁₋₆SO₃H H T8-28 SO₃H SO₃H H H CONH—Y(CH₂)₁₋₆CO—Mal CONH-Z (CH₂)₁₋₆SO₃H H T8-29 H H SO₃H SO₃H CONH—Y(CH₂)₁₋₆CO—Mal CONH-Z (CH₂)₁₋₆SO₃H H T8-30 SO₃H SO₃H SO₃H SO₃H CONH—Y(CH₂)₁₋₆CO—Mal CONH-Z (CH₂)₁₋₆SO₃H H OSu = N-Hydroxysuccinimidyl; MAL =N-(2-(N-maleimido)-ethyl)-amide

TABLE 9 Examplary compounds according to this disclosure.

No R₂ R₃ R₄ R₅ R₆ R₇ Q T9-1 SO₃H SO₃H SO₃H H SO₃H H H H Cl T9-2 SO₃HSO₃H H H H H H H Cl T9-3 SO₃H SO₃H SO₃H SO₃H H H H H Cl T9-4 SO₃H SO₃H HH SO₃H SO₃H H H Cl T9-5 SO₃H SO₃H SO₃H SO₃H SO₃H SO₃H H H Cl T9-6 SO₃HSO₃H SO₃H H SO₃H H H H S(CH₂)₁₋₆COOH T9-7 SO₃H SO₃H H H H H H HS(CH₂)₁₋₆COOH T9-8 SO₃H SO₃H SO₃H SO₃H H H H H S(CH₂)₁₋₆COOH T9-9 SO₃HSO₃H H H SO₃H SO₃H H H S(CH₂)₁₋₆COOH T9-10 SO₃H SO₃H SO₃H SO₃H SO₃H SO₃HH H S(CH₂)₁₋₆COOH T9-11 SO₃H SO₃H SO₃H H SO₃H H H H S(CH₂)₁₋₆CO—OSuT9-12 SO₃H SO₃H H H H H H H S(CH₂)₁₋₆CO—OSu T9-13 SO₃H SO₃H SO₃H SO₃H HH H H S(CH₂)₁₋₆CO—OSu T9-14 SO₃H SO₃H H H SO₃H SO₃H H H S(CH₂)₁₋₆CO—OSuT9-15 SO₃H SO₃H SO₃H SO₃H SO₃H SO₃H H H S(CH₂)₁₋₆CO—OSu T9-16 SO₃H SO₃HSO₃H H SO₃H H H H S(CH₂)₁₋₆CO—MAL T9-17 SO₃H SO₃H H H H H H HS(CH₂)₁₋₆CO—MAL T9-18 SO₃H SO₃H SO₃H SO₃H H H H H S(CH₂)₁₋₆CO—MAL T9-19SO₃H SO₃H H H SO₃H SO₃H H H S(CH₂)₁₋₆CO—MAL T9-20 SO₃H SO₃H SO₃H SO₃HSO₃H SO₃H H H S(CH₂)₁₋₆CO—MAL T9-21 SO₃H SO₃H SO₃H H SO₃H H H HS-Ph-(CH₂)₀₋₆COOH T9-22 SO₃H SO₃H H H H H H H S-Ph-(CH₂)₀₋₆COOH T9-23SO₃H SO₃H SO₃H SO₃H H H H H S-Ph-(CH₂)₀₋₆COOH T9-24 SO₃H SO₃H H H SO₃HSO₃H H H S-Ph-(CH₂)₀₋₆COOH T9-25 SO₃H SO₃H SO₃H SO₃H SO₃H SO₃H H HS-Ph-(CH₂)₀₋₆COOH T9-26 SO₃H SO₃H SO₃H H SO₃H H H H S-Ph-(CH₂)₀₋₆CO—OSuT9-27 SO₃H SO₃H H H H H H H S-Ph-(CH₂)₀₋₆CO—OSu T9-28 SO₃H SO₃H SO₃HSO₃H H H H H S-Ph-(CH₂)₀₋₆CO—OSu T9-29 SO₃H SO₃H H H SO₃H SO₃H H HS-Ph-(CH₂)₀₋₆CO—OSu T9-30 SO₃H SO₃H SO₃H SO₃H SO₃H SO₃H H HS-Ph-(CH₂)₀₋₆CO—OSu T9-31 SO₃H SO₃H SO₃H H SO₃H H H HS-Ph-(CH₂)₀₋₆CO—MAL T9-32 SO₃H SO₃H H H H H H H S-Ph-(CH₂)₀₋₆CO—MALT9-33 SO₃H SO₃H SO₃H SO₃H H H H H S-Ph-(CH₂)₀₋₆CO—MAL T9-34 SO₃H SO₃H HH SO₃H SO₃H H H S-Ph-(CH₂)₀₋₆CO—MAL T9-35 SO₃H SO₃H SO₃H SO₃H SO₃H SO₃HH H S-Ph-(CH₂)₀₋₆CO—MAL T9-36 SO₃H SO₃H SO₃H H SO₃H H H HO-Ph-(CH₂)₀₋₆COOH T9-37 SO₃H SO₃H H H H H H H O-Ph-(CH₂)₀₋₆COOH T9-38SO₃H SO₃H SO₃H SO₃H H H H H O-Ph-(CH₂)₀₋₆COOH T9-39 SO₃H SO₃H H H SO₃HSO₃H H H O-Ph-(CH₂)₀₋₆COOH T9-40 SO₃H SO₃H SO₃H SO₃H SO₃H SO₃H H HO-Ph-(CH₂)₀₋₆COOH T9-41 SO₃H SO₃H SO₃H H SO₃H H H H O-Ph-(CH₂)₀₋₆CO—OSuT9-42 SO₃H SO₃H H H H H H H O-Ph-(CH₂)₀₋₆CO—OSu T9-43 SO₃H SO₃H SO₃HSO₃H H H H H O-Ph-(CH₂)₀₋₆CO—OSu T9-44 SO₃H SO₃H SO₃H SO₃H H H H HO-Ph-(CH₂)₀₋₆CO—OSu T9-45 SO₃H SO₃H SO₃H SO₃H SO₃H SO₃H H HO-Ph-(CH₂)₀₋₆CO—OSu T9-46 SO₃H SO₃H SO₃H H SO₃H H H HO-Ph-(CH₂)₀₋₆CO—MAL T9-47 SO₃H SO₃H H H H H H H O-Ph-(CH₂)₀₋₆CO—MALT9-48 SO₃H SO₃H SO₃H SO₃H H H H H O-Ph-(CH₂)₀₋₆CO—MAL T9-49 SO₃H SO₃H HH SO₃H SO₃H H H O-Ph-(CH₂)₀₋₆CO—MAL T9-50 SO₃H SO₃H SO₃H SO₃H SO₃H SO₃HH H O-Ph-(CH₂)₀₋₆CO—MAL OSu = N-Hydroxysuccinimidyl; MAL =N-(2-(N-maleimido)-ethyl)-amide

TABLE 10 Examplary compounds according to this disclosure.

No R₂ R₃ R₄ R₅ Q T10-1 SO₃H H SO₃H H Cl T10-2 H H SO₃H SO₃H Cl T10-3SO₃H SO₃H H H Cl T10-4 SO₃H SO₃H SO₃H SO₃H Cl T10-5 H SO₃H H SO₃H ClT10-6 SO₃H H SO₃H H S(CH₂)₁₋₆COOH T10-7 H H SO₃H SO₃H S(CH₂)₁₋₆COOHT10-8 SO₃H SO₃H H H S(CH₂)₁₋₆COOH T10-9 SO₃H SO₃H SO₃H SO₃HS(CH₂)₁₋₆COOH T10-10 H SO₃H H SO₃H S(CH₂)₁₋₆COOH T10-11 SO₃H H SO₃H HS(CH₂)₁₋₆CO—OSU T10-12 H H SO₃H SO₃H S(CH₂)₁₋₆CO—OSU T10-13 SO₃H SO₃H HH S(CH₂)₁₋₆CO—OSU T10-14 SO₃H SO₃H SO₃H SO₃H S(CH₂)₁₋₆CO—OSU T10-15 HSO₃H H SO₃H S(CH₂)₁₋₆CO—OSU T10-16 SO₃H H SO₃H H S(CH₂)₁₋₆CO—MAL T10-17H H SO₃H SO₃H S(CH₂)₁₋₆CO—MAL T10-18 SO₃H SO₃H H H S(CH₂)₁₋₆CO—MALT10-19 SO₃H SO₃H SO₃H SO₃H S(CH₂)₁₋₆CO—MAL T10-20 H SO₃H H SO₃HS(CH₂)₁₋₆CO—MAL T10-21 SO₃H H SO₃H H SPh(CH₂)₀₋₆COOH T10-22 H H SO₃HSO₃H Sph(CH₂)₀₋₆COOH T10-23 SO₃H SO₃H H H SPh(CH₂)₀₋₆COOH T10-24 SO₃HSO₃H SO₃H SO₃H SPh(CH₂)₀₋₆COOH T10-25 H SO₃H H SO₃H SPh(CH₂)₀₋₆COOHT10-26 SO₃H H SO₃H H O-Ph(CH₂)₀₋₆COOH T10-27 H H SO₃H SO₃HO-Ph(CH₂)₀₋₆COOH T10-28 SO₃H SO₃H H H O-Ph(CH₂)₀₋₆COOH T10-29 SO₃H SO₃HSO₃H SO₃H O-Ph(CH₂)₀₋₆COOH T10-30 H SO₃H H SO₃H O-Ph(CH₂)₀₋₆COOH T10-31SO₃H H SO₃H H SPh(CH₂)₀₋₆CO—OSU T10-32 H H SO₃H SO₃H SPh(CH₂)₀₋₆CO—OSUT10-33 SO₃H SO₃H H H SPh(CH₂)₀₋₆CO—OSU T10-34 SO₃H SO₃H SO₃H SO₃HSPh(CH₂)₀₋₆CO—OSU T10-35 H SO₃H H SO₃H SPh(CH₂)₀₋₆CO—OSU T10-36 SO₃H HSO₃H H O-Ph(CH₂)₀₋₆CO—OSU T10-37 H H SO₃H SO₃H O-Ph(CH₂)₀₋₆CO—OSU T10-38SO₃H SO₃H H H O-Ph(CH₂)₀₋₆CO—OSU T10-39 SO₃H SO₃H SO₃H SO₃HO-Ph(CH₂)₀₋₆CO—OSU T10-40 H SO₃H H SO₃H O-Ph(CH₂)₀₋₆CO—OSU T10-41 SO₃H HSO₃H H SPh(CH₂)₀₋₆CO—MAL T10-42 H H SO₃H SO₃H SPh(CH₂)₀₋₆CO—MAL T10-43SO₃H SO₃H H H SPh(CH₂)₀₋₆CO—MAL T10-44 SO₃H SO₃H SO₃H SO₃HSPh(CH₂)₀₋₆CO—MAL T10-45 H SO₃H H SO₃H SPh(CH₂)₀₋₆CO—MAL T10-46 SO₃H HSO₃H H O-Ph(CH₂)₀₋₆CO—MAL T10-47 H H SO₃H SO₃H O-Ph(CH₂)₀₋₆CO—MAL T10-48SO₃H SO₃H H H O-Ph(CH₂)₀₋₆CO—MAL T10-49 SO₃H SO₃H SO₃H SO₃HO-Ph(CH₂)₀₋₆CO—MAL T10-50 H SO₃H H SO₃H O-Ph(CH₂)₀₋₆CO—MAL OSu =N-Hydroxysuccinimidyl; MAL = N-(2-(N-maleimido)-ethyl)-amide

III. Synthesis

Also provided are methods for the synthesis of fluorochromes and keyintermediates (bridges, B, and functionalized polycyclic compounds P₁ orP₂). In particular, methods are described that allow the use offunctional groups that impart water solubility to the resultingfluorochrome, but present significant challenges during synthesis due toincompatibility with solvents or reagents required. The new proceduresallow synthesis of commercially useful quantities of the intermediatesrequired to make the fluorochromes of the present disclosure.

Generally, the fluorochrome compounds of the present disclosure can besynthesized in the following manner. Two equivalents of a suitablysubstituted heterocyclic moiety (P₁ and P₂, wherein P₁═P₂) are reactedwith a bridge precursor B bearing two reactive aldehyde groups, eachprotected in the form of an imine, such as an anilinium imine, in amixture of acetic acid, acetic anhydride with the addition of a suitablebase, such as potassium acetate, at a temperature of about 100° C. forabout two hours to form a symmetrical fluorochrome, followed bypurification by HPLC as shown in the exemplary scheme below.

In certain embodiments, the fluorochrome compounds are unsymmetricaloptionally where P₁ is not identical to P₂. Such unsymmetricalfluorochrome compounds can be synthesized as follows. One equivalent ofa suitably substituted heterocyclic moiety (P₁, wherein P₁≠P₂) isreacted with a bridge precursor B bearing two reactive aldehyde groups,each protected in the form of an imine, such as an anilinium imine, in amixture of acetic acid, acetic anhydride in the absence of base, at atemperature of about 100° C. for about two hours to form amono-substituted bridge intermediate (I), after which one equivalent ofa different suitably substituted heterocyclic moiety (P₂) is added alongwith a suitable base, such as potassium acetate, and heating to 100° C.is resumed for about two hours to form the unsymmetrical fluorochrome,which can be purified by HPLC, according to the following scheme:

In some embodiments, a suitably substituted bridge B can further reactwith other molecules to form a linker L. As an illustration, a linkersubstitution can be performed on a chloride substituted bridge asfollows. One equivalent of chloro-substituted bridge fluorochrome isreacted with 2 equivalents of 2-(4-mercaptophenyl) acetic acid in DMFwith 2% pyridine for 15 minutes at room temperature. The resultinglinker (L) substituted fluorochrome is then purified by HPLC.

In some embodiments, methods are provided for the synthesis of suitablysubstituted aromatic heterocycles, such as benz[c,d]indolium,thiopyrylium or acridinium salts. Optionally, sulfonate groups areintroduced to a suitably activated benz[c,d]indolinone, acridone, orthiopyranone, through reaction with chlorosulfonic acid followed byhydrolysis and conversion to the to the corresponding benz[c,d]indolium,thiopyrylium or acridinium salts.

In some embodiments, the starting material is benz[c,d]indolin-2-one.

In some embodiments, the starting material is2,6-diphenylthiopyran-4-one.

In some embodiments, the starting material is 10-methylacridin-9-one.

Dyes having absorption max in the SWIR window (1000-1300 nm) aresuitable for exciting with 980 nm laser. However, existing dyes are verydifficult to solubilize in aqueous or most organic solvents, and thusare practically not suitable for use in biological applications. Assuch, these molecules are optionally excluded from the presentdisclosure. Lack of a functional group for conjugating to other moietiesand lack of polar substituents that enable aqueous solubility furthercomplicate their direct use in any biologically important detectionsystems.

Addressing this issue, some methods as provided herein may be used tosynthesize heterocycles bearing one or more aqueous soluble substituentssuch as sulfonates or “sulfonic acid” groups as in the table below forsynthesizing water soluble SWIR dyes; wherein, R is an alklyl-X (withX═H or SO₃H or COOH or NH₂, or OH or halide), and one or more of R₁, R₂,and R₃ being a SO₃ ⁻ or SO₃H group.

ACR-1 TPS-1 TPS-2 TPS-2 DSB[cd]Indole

Alternatively, provided herein are compounds that may be used tosynthesize water soluble SWIR dyes as provided herein. Optionally,compounds as provided herein may be represented by one of the followingstructures:

or a salt thereof, wherein:

R₁ is independently for each occurrence, hydrogen substituted orunsubstituted C₁ to C₂₄ alkyl, substituted or unsubstituted alkylaryl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl, sulfonate, aryl sulfonate, alkyl sulfonate,taurine, carboxylate, amine, alkylamine, arylamine, alkylammonium,arylammonium, sulfonamide, halogen, hydroxy, amide, nitro, cyano, azide,O-alkyl, S-alkyl, silyl, trialkylsilyl, O-silyl, haloalkyl,alkylsulfhydryl, trifluoromethyl, hydrazide, substituted orunsubstituted aryl, heteroaryl, or heterocyclic (e.g. morpholine)alkynyl, carboxyalkyl, aminoalkyl, haloalkyl, azidoalkyl, amide, aminoacid, or peptide or L;

L is absent or is a linker moiety, optionally bearing a functional groupor reactive group, wherein said functional group or reactive group is acarboxylate, carboxyalkyl, maleimide, succinimidyl ester, carboxamide,propargyl, azidoalkyl, alkyne, isothiocyanate, of —NH₂—OH, —SH, —SO₃H,carboxyl, —COCl, —CONHNH₂, acetoxymethyl esters, substituted andunsubstituted N-hydroxysuccinimidyl esters, substituted andunsubstituted N-hydroxysulfosuccinimido esters, nitro- or fluoro orphenol esters, azide, —COCH₂I, phosphoramidite, phthalamido, acylfluoride, acyl chloride, acyl azide, tyramide, cinnamamide,hydroxycinnamamide, aldehyde, ketone, phosphoramidite, isocyanate,isothiocyanate, sulfonyl chloride, maleimide or biotin;

R₂ is a substituted or unsubstituted C₁ to C₂₄ alkyl, substituted orunsubstituted alkylaryl, substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl, substituted or unsubstituted alkenyl,substituted or unsubstituted alkynyl, aryl sulfonate, C₁ to C₂₄ alkylsulfonate, C₁-C₂₄ alkyl carboxylate, aryl carboxylate, C₁-C₂₄alkylamine, arylamine, C₁-C₂₄ alkylammonium, arylammonium, orsubstituted or unsubstituted polyethylene glycol.

In some aspects, R₂ is a substituted or unsubstituted C₁ to C₂₄ alkyl,substituted or unsubstituted alkylaryl, substituted or unsubstitutedaryl, aryl sulfonate, C₁ to C₂₄ alkyl sulfonate, C₁-C₂₄ alkylcarboxylate, or aryl carboxylate. Optionally, R₂ is a C₁-C₂₄ alkylcarboxylate optionally a C₂ alkyl carboxylate.

Optionally, R₁ is a sulfonate, alkyl sulfonate, arylsulfonate ortaurine. In some aspects, in addition to R₁ being a sulfonate, alkylsulfonate, arylsulfonate or taurine, R₂ is optionally a substituted orunsubstituted C₁ to C₂₄ alkyl, substituted or unsubstituted alkylaryl,substituted or unsubstituted aryl, aryl sulfonate, C₁ to C₂₄ alkylsulfonate, C₁-C₂₄ alkyl carboxylate, or aryl carboxylate.

Any of the foregoing may be used as precursors for one or more of thefluorescent compounds as provided herein.

IV. Properties and Uses

Fluorochrome compounds with low solubility in water frequently undergoaggregation or conformationally induced hypsochromic shifts in opticalproperties. In some embodiments, the fluorochrome compounds aresolubilized by incorporation of one or more solubilizing groups. Thesolubilizing groups are optionally sulfonates. Optionally, the watersoluble fluorochrome compounds substantially retain their SWIR opticalproperties when in solution in water without undergoing a hypsochromicshift in absorbance or emission relative to the optical properties in anorganic solvent such as methanol. As used herein, the term water solublewith respect to SWIR dyes means full soluble in water to a concentrationof 1 μM or greater, optionally 10 μM or greater, optionally to 100 μM.

It is contemplated that the fluorochrome compounds as provided hereinare particularly useful for photon mediated imaging and detectionapplications. In some aspects, the fluorochrome compounds are linked,conjugated or otherwise bound to targeting ligands, such as peptides,drugs, proteins, antibodies or nucleic acids that bind with specificityto a particular biological target, for example in a biological sample,on a cell, on a tissue, or in a living animal or patient. In this way,the SWIR optical properties can be used to detect, visualize, or imagethe presence, absence or spatial distribution of the biological targetin the sample, cell, tissue, animal or patient.

In other aspects, the fluorochrome compounds are linked, conjugated orotherwise bound to one or more carrier molecules, illustratively but notlimited to 3,3-diphenylpropylamine, pamidronate, alendronate,bisphosphonates, polyethylene glycol, polyvinylpyrrolidone,N-(2-hydroxypropyl)methacrylamide, polymers, copolymers, proteins,protamine, poly-L-arginine, poly-D-arginine, liposomes, ornanoparticles. In other aspects, the conjugates of the fluorochromecompounds direct the localization of the fluorochrome compounds incells, tissues, animals or patients, to, for example, a cell nucleus,cytoplasm, lysosomes, mitochondria, vasculature, bone, lung, kidney,liver, heart, brain, or tumor tissue.

In some aspects, the fluorochrome compounds are useful for microscopy,flow cytometry, or tissue imaging.

In some aspects, the fluorochrome compounds are useful for in vivofluorescence optical imaging.

In some aspects the fluorochrome compounds are useful for ex vivofluorescence optical imaging.

In some aspects, the fluorochrome compounds are useful for photoacousticimaging.

In some aspects, the fluorochrome compounds act as photosensitizers togenerate singlet oxygen upon excitation with light. Optionally,fluorochrome compounds are useful for photodynamic therapy. Optionally,fluorochrome compounds may be used as a sensitizer in a luminescentsinglet oxygen channeling assay.

Another aspect of this disclosure includes a peptide, protein or otherbiomolecule that presents a proteolytic or enzymolytic scissile bond,and two or more fluorochrome compounds as provided herein that arechemically linked to the peptide, protein or biomolecule such that theirfluorescence is significantly quenched. Upon the action of an enzyme bye.g. enzymatic cleavage upon the peptide, protein or biomoleculescissile bond, the fluorochrome compounds are separated and the agentemits a fluorescent signal when excited by electromagnetic radiation ofappropriate wavelength and frequency. As used herein, the term“quenched” is understood to mean the process of partially or completelyreducing the fluorescent signal from a fluorophore. For example, afluorescent signal from the fluorochrome compound can be reduced inter-or intra-molecularly through the placement of a second fluorochrome(either the same or a different compound) in close proximity to thefirst fluorochrome or the placement of a non-fluorogenic quenchingchromophore molecule, e.g., quencher, in close proximity to the firstfluorophore. The agent is de-quenched (or activated), for example,through the enzymatic cleavage of a peptide, protein or biomoleculeproteolytic or enzymolytic scissile bond.

In some embodiments, the fluorochrome compounds may have very lowintrinsic fluorescence (quantum yield less than about 0.01%) but retainhigh absorption (molar extinction coefficient higher than about 50,000M⁻¹cm⁻¹) in the NIR to SWIR region of the electromagnetic spectrum. Itis contemplated that such fluorochrome compounds could be used asquencher compounds when in close proximity to another fluorescentcompound that emits fluorescence at wavelengths close to the absorptionwavelengths. Such compounds, containing one fluorescent compound and acomplementary quencher compound with low intrinsic fluorescence could beactivatable if, for example, the fluorescent compound and the quenchercompound are separated by a peptide, protein or biomolecule enzymolyticscissile bond that is recognized and cleaved by a particular enzyme orprotease. It is further contemplated that the intramolecularly quenchedfluorochrome and quencher compounds could be activated through chemicalmeans as well, such as an oxidation or reduction with or without the aidof an enzyme.

(a) Imaging Methods

The present disclosure provides methods for in vitro and in vivo imagingusing the compounds disclosed herein. For a review of optical imagingtechniques, see, e.g., Alfano et al., ANN. NY ACAD. SCI. 820:248-270(1997); Weissleder, Nature Biotechnology 19, 316-317 (2001);Ntziachristos et al., Eur. Radiol. 13:195-208 (2003); Graves et al.,Curr. Mol. Med. 4:419-430 (2004); Citrin et al., Expert Rev. AnticancerTher. 4:857-864 (2004); Ntziachristos, Ann. Rev. Biomed. Eng. 8:1-33(2006); Koo et al., Cell Oncol. 28:127-139 (2006); and Rao et al., Curr.Opin. Biotechnol. 18:17-25 (2007).

Optical imaging includes all methods from direct visualization withoutuse of any device and use of devices such as various scopes, cathetersand optical imaging equipment, for example computer based hardware fortomographic presentations. The imaging agents are useful with opticalimaging modalities and measurement techniques including, but not limitedto: endoscopy; fluorescence endoscopy; luminescence imaging; timeresolved transmittance imaging; transmittance imaging; nonlinearmicroscopy; confocal imaging; acousto-optical imaging; photoacousticimaging; reflectance spectroscopy; spectroscopy; coherenceinterferometry; interferometry; optical coherence tomography; diffuseoptical tomography and fluorescence mediated molecular tomography(continuous wave, time domain frequency domain systems and earlyphoton), and measurement of light scattering, absorption, polarization,luminescence, fluorescence lifetime, quantum yield, and quenching.

An imaging system useful in the practice of the imaging methods asprovided herein typically includes three basic components: (1) anappropriate light source for inducing excitation of the compound, (2) asystem for separating or distinguishing emissions from light used forfluorophore excitation, and (3) a detection system. The detection systemcan be hand-held or incorporated into other useful imaging devices, suchas intraoperative microscopes. Exemplary detection systems include anendoscope, catheter, tomographic system, hand-held imaging system, or aintraoperative microscope.

Optionally, the light source provides monochromatic (or substantiallymonochromatic) light. The light source can be a suitably filtered light,i.e., bandpass light from a broadband source. For example, light from a150-watt halogen lamp can be passed through a suitable bandpass filtercommercially available from Omega Optical (Brattleboro, Vt.). Dependingupon the system, the light source can be a laser. See, e.g., Boas etal., PROC. NATL. ACAD. SCI. USA 91:4887-4891, 1994; Ntziachristos etal., PROC. NATL. ACAD. SCI. USA 97:2767-2772, 2000; and Alexander, J.CLIN. LASER MED. SURG. 9:416-418, 1991. Information on lasers forimaging can be found, for example, at Imaging Diagnostic Systems, Inc.,Plantation, Fla. and various other sources. A high pass or bandpassfilter can be used to separate optical emissions from excitation light.A suitable high pass or bandpass filter is commercially available fromOmega Optical, Burlington, Vt.

In general, the light detection system can be viewed as including alight gathering/image forming component and a light/signaldetection/image recording component. Although the light detection systemcan be a single integrated device that incorporates both components, thelight gathering/image forming component and light detection/imagerecording component are discussed separately.

A particularly useful light gathering/image forming component is anendoscope. Endoscopic devices and techniques which have been used for invivo optical imaging of numerous tissues and organs, includingperitoneum (Gahlen et al., J. PHOTOCHEM. PHOTOBIOL. B 52:131-135, 1999),ovarian cancer (Major et al., Gynecol. Oncol. 66:122-132, 1997), colonand rectum (Mycek et al., GASTROINTEST. ENDOSC. 48:390-394, 1998; andStepp et al., ENDOSCOPY 30:379-386, 1998), bile ducts (Izuishi et al.,HEPATOGASTROENTEROLOGY 46:804-807, 1999), stomach (Abe et al., ENDOSCOPY32:281-286, 2000), bladder (Kriegmair et al., UROL. INT. 63:27-31, 1999;and Riedl et al., J. ENDOUROL. 13:755-759, 1999), lung (Hirsch et al.,CLIN CANCER RES 7:5-220, 2001), brain (Ward, J. LASER APPL. 10:224-228,1998), esophagus, and head and neck regions can be employed.

Other types of light gathering components are catheter-based devices,including fiber optics devices. Such devices are particularly suitablefor intravascular imaging. See, e.g., Tearney et al., SCIENCE276:2037-2039, 1997; and CIRCULATION 94:3013, 1996.

Still other imaging technologies, including phased array technology(Boas et al., PROC. NATL. ACAD. SCI. USA 91:4887-4891, 1994; Chance,ANN. NY ACAD. SCI. 838:29-45, 1998), optical tomography (Cheng et al.,OPTICS EXPRESS 3:118-123, 1998; and Siegel et al., OPTICS EXPRESS4:287-298, 1999), intravital microscopy (Dellian et al., BR. J. CANCER82:1513-1518, 2000; Monsky et al., CANCER RES. 59:4129-4135, 1999; andFukumura et al., CELL 94:715-725, 1998), confocal imaging (Korlach etal., PROC. NATL. ACAD. SCI. USA 96:8461-8466, 1999; Rajadhyaksha et al.,J. INVEST. DERMATOL. 104:946-952, 1995; and Gonzalez et al., J. MED.30:337-356, 1999) and fluorescence molecular tomography (FMT)(Nziachristos et al., NATURE MEDICINE 8:757-760, 2002; U.S. Pat. No.6,615,063, PCT WO 03/102558, and PCT WO 03/079015) can be used with theimaging agents of this disclosure. Similarly, the imaging agents can beused in a variety of imaging systems, for example, (1) the IVIS® ImagingSystems: 100 Series, 200 Series (Xenogen, Alameda, Calif.), (2) SPECTRUMand LUMINA (Xenogen, Alameda, Calif.), (3) the SoftScan® or the eXploreOptix™ (GE Healthcare, United Kingdom), (4) Maestro™ and Nuance™-2Systems (CRi, Woburn, Mass.), (5) Image Station In-Vivo FX fromCarestream Molecular Imaging, Rochester, N.Y. (formerly Kodak MolecularImaging Systems), (6) OV100, IV100 (Olympus Corporation, Japan), (7)Cellvizio Mauna Kea Technologies, France), (8)] NanoSPECT/CT or HiSPECT(Bioscan, Washington, D.C.), (9) CTLM® or LILA™ (Imaging DiagnosticSystems, Plantation, Fla.), (10) DYNOT™ (NIRx Medical Technologies, GlenHead, N.Y.), and (11) NightOWL Imaging Systems by Berthold Technologies,Germany.

A variety of light detection/image recording components, e.g., chargecoupled device (CCD) systems or photographic film, can be used in suchsystems. The choice of light detection/image recording depends onfactors including the type of light gathering/image forming componentbeing used. It is understood, however, that the selection of suitablecomponents, assembling them into an optical imaging system, andoperating the system is within ordinary skill in the art.

(i) In Vivo Imaging Methods

With respect to optical in vivo imaging, such a method comprises (a)administering to a subject one or more of the compounds as describedherein, (b) allowing sufficient time to permit the compound todistribute or localize within the subject, and (c) detecting a signalemitted by the compound. The signal emitted by the compound can be usedto construct an image, for example, a tomographic image. The foregoingsteps can be repeated at predetermined time intervals thereby to permitevaluation of the emitted signals of the imaging compounds in thesubject over time.

In another in vivo imaging method, the method comprises the steps of (a)administering to a subject one or more of the compounds described hereinthat contains a fluorochrome; (b) allowing sufficient time to permit thecompound to distribute or localize within the subject; (c) exposing thesubject to light of a wavelength absorbable by the fluorochrome, and (d)detecting a signal emitted by the compound. The foregoing steps can berepeated at predetermined time intervals thereby to permit evaluation ofthe emitted signals of the compounds in the subject over time. Theilluminating and/or detecting steps (steps (c) and (d), respectively)can be performed using an endoscope, catheter, tomographic system,planar system, hand-held imaging system, goggles, an intraoperativemicroscope, or other suitable device.

Before or during the method steps, a detection system can be positionedaround or in the vicinity of a subject (for example, an animal (e.g.,human) to detect signals emitted from the subject. The emitted signalscan be processed to construct an image, for example, a tomographicimage. In addition, the processed signals can be displayed as imageseither alone or as fused (combined) images.

In addition, it is possible to practice an in vivo imaging method thatselectively detects and images one, two or more molecular imagingprobes, optionally including the currently provided compoundssimultaneously. In such an approach, for example, in step (a) notedabove, two or more imaging probes whose signal properties aredistinguishable from one another are administered to the subject, eitherat the same time or sequentially, wherein at least one of the molecularimaging probes is a compound as provided herein. The use of multipleprobes permits the recording of multiple biological processes, functionsor targets.

The subject may be a vertebrate, for example, a mammal, for example, ahuman. The subject may also be a non-vertebrate (for example, C.elegans, drosophila, or another model research organism, etc.) used inlaboratory research.

Information provided by such in vivo imaging approaches, for example,the presence, absence, or level of emitted signal can optionally be usedto detect and/or monitor a health condition in the subject. Exemplaryhealth conditions include, without limitation, autoimmune disease, bonedisease, cancer, cardiovascular disease, environmental disease,dermatological disease, immunologic disease, inherited disease,infectious disease, metabolic disease, neurodegenerative disease,ophthalmic disease, respiratory disease, early stages of such diseases,and progression or remission of disease (e.g. tumor size changes). Inaddition, in vivo imaging can be used to assess the effect of a compoundor therapy by using the imaging agents, wherein the subject is imagedprior to and after treatment with the compound or therapy, and thecorresponding signal/images are compared.

The compounds as provided herein also can be used in in vivo imagingmethod where cells labeled with the compound are administered to therecipient. The cells can be labeled with the compounds either in vivo orex vivo. In the ex vivo approach, cells can be derived directly from asubject or from another source (e.g., from another subject, cellculture, etc.). The compounds can be mixed with the cells to effectivelylabel the cells and the resulting labeled cells administered to thesubject into a subject in step (a). Any of steps (b)-(d) then arefollowed as described above. This method can be used for monitoringtrafficking and localization of certain cell types, including T-cells,tumor cells, immune cells and stem cells, and other cell types. Inparticular, this method may be used to monitor cell-based therapies.

It is understood that the formulation of the compounds, the choice ofmode of administration, the dosages of compounds administered to thesubject, and the timing between administration of the imaging compoundsand imaging may be selected by one of ordinary skill in the art.

The foregoing methods can be used to determine a number of indicia,optionally including tracking the localization of the imagingcompound(s) in the subject over time or assessing changes or alterationsin the metabolism and/or excretion of the imaging compounds in thesubject over time. The methods can also be used to follow therapy forhealth conditions by imaging molecular events and biological pathwaysmodulated by such therapy, including but not limited to determiningefficacy, optimal timing, optimal dosing levels (including forindividual patients or test subjects), and synergistic effects ofcombinations of therapy.

The methods and compositions of this disclosure can be used to help aphysician or surgeon to identify and characterize areas of interest ordisease, such as arthritis, cancers and specifically colon polyps, orvulnerable or unstable plaque, to distinguish diseased and normaltissue, such as detecting tumor margins that are difficult to detectusing an ordinary operating microscope, e.g., in brain surgery, to helpdictate a therapeutic or surgical intervention, e.g., by determiningwhether a lesion is cancerous and should be removed or non-cancerous andleft alone, or in surgically staging a disease, e.g., intraoperativelymph node staging, sentinel lymph node mapping, or assessingintraoperative bleeding.

The methods and compositions of this disclosure can also be used in thedetection, characterization and/or determination of the localization ofa health condition, especially early disease, the severity of a diseaseor a disease-associated condition, the staging of a disease, and/ormonitoring a disease. The presence, absence, or level of an emittedsignal can be indicative of a disease state.

The methods and compositions of this disclosure can also be used tomonitor and/or guide various therapeutic interventions, such as surgicalprocedures, and monitoring drug therapy, including cell based therapies.The methods of this disclosure can also be used in prognosis of a healthcondition.

With respect to each of the foregoing, examples of such healthconditions that can be detected or monitored (before, during or aftertherapy) include but are not limited to inflammation (for example,inflammation caused by arthritis, for example, rheumatoid arthritis),cancer (for example, colorectal, ovarian, lung, breast, prostate,cervical, testicular, skin, brain, gastrointestinal, pancreatic, liver,kidney, bladder, stomach, leukemia, mouth, esophageal, bone),cardiovascular disease (for example, atherosclerosis and inflammatoryconditions of blood vessels, ischemia, stroke, thrombosis, disseminatedintravascular coagulation), dermatologic disease (for example, Kaposi'sSarcoma, psoriasis, allergic dermatitis), ophthalmic disease (forexample, macular degeneration, diabetic retinopathy), infectious disease(for example, bacterial, viral, fungal and parasitic infections,including Acquired Immunodeficiency Syndrome, malaria, Chagas Disease,Schistosomiasis), immunologic disease (for example, an autoimmunedisorder, lymphoma, multiple sclerosis, rheumatoid arthritis, diabetesmellitus, lupus erythematosis, myasthenia gravis, Graves disease),central nervous system disease (for example, a neurodegenerativedisease, such as Parkinson's disease or Alzheimer's disease,Huntington's Disease, amyotrophic lateral sclerosis, prion disease),inherited diseases, metabolic diseases, environmental diseases (forexample, lead, mercury and radioactive poisoning, skin cancer),bone-related disease (for example, osteoporosis, primary and metastaticbone tumors, osteoarthritis), neurodegenerative disease, andsurgery-related complications (such as graft rejection, organ rejection,alterations in wound healing, fibrosis or other complications related tosurgical implants).

The methods and compositions described herein can, therefore, be used,for example, to determine the presence and/or localization of tumorcells, the presence and/or localization of inflammation, including thepresence of activated macrophages, for instance in atherosclerosis orarthritis, the presence and in localization of vascular diseaseincluding areas at risk for acute occlusion (i.e., vulnerable plaques)in coronary and peripheral arteries, regions of expanding aneurysms,unstable plaque in carotid arteries, and ischemic areas. The methods andcompositions of this disclosure can also be used in identification andevaluation of cell death, injury, apoptosis, necrosis, hypoxia andangiogenesis. The methods and compositions can also be used for drugdelivery and to monitor drug delivery, especially when drugs ordrug-like molecules are chemically attached to one or more of thecompounds as provided herein. Exemplary drug molecules includechemotherapeutic and cytostatic agents and photodynamic agents includingbut not limited to porfimer sodium, motexafin lutetium, cetirizinedihydrochloride, aminolevulinic acid, hypericin, benzoporphyrinderivative, and porphyrins.

In addition, the methods and compositions described herein can be usedto image angiogenesis (new blood vessel formation) in a subject. Themethod comprises administering to a subject (for example, a human oranimal) an amount of one or more of the imaging compounds describedherein sufficient to facilitate angiogenesis imaging. After sufficienttime to permit the agent to distribute within the animal or distributewithin the area to be imaged, the presence and/or amount of the agent isdetermined. The presence and/or amount of the agent can then be used tocreate an image, for example, a tomographic image, representative of newblood vessel formation in the subject.

(ii) In Vitro Imaging Methods

With respect to in vitro imaging, the imaging compounds can be used in avariety of in vitro assays. For example, an exemplary in vitro imagingmethod comprises: (a) contacting a sample, for example, a biologicalsample, with one or more of the imaging compounds described herein; (b)allowing the agent(s) to interact with a biological target in thesample; (c) optionally, removing unbound agent; and (d) detecting asignal emitted from the agent thereby to determine whether the imagingcompounds has been activated by or bound to the biological target. Whenthe imaging compound comprises a fluorochrome, step (d) may furtherinclude illuminating the sample with light of a wavelength absorbable bythe fluorochrome to produce the emitted signal.

After an imaging compound has been designed, synthesized, and optionallyformulated, it can be tested in vitro to assess its biological andperformance characteristics. For instance, different types of cellsgrown in culture can be used to assess the biological and performancecharacteristics of the imaging compound. Cellular uptake, binding orcellular localization of the agent can be assessed using techniquesknown in the art, including, for example, fluorescent microscopy, FACSanalysis, immunohistochemistry, immunoprecipitation, in situhybridization and Forster resonance energy transfer (FRET) orfluorescence resonance energy transfer. By way of example, the imagingcompounds can be contacted with a sample for a period of time and thenwashed to remove any free imaging compounds. The sample can then beviewed using an appropriate detection device such as a fluorescentmicroscope equipped with appropriate filters matched to the opticalproperties of a fluorescent agent. Fluorescence microscopy of cells inculture or scintillation counting is also a convenient means fordetermining whether uptake and binding has occurred. Tissues, tissuesections and other types of samples such as cytospin samples can also beused in a similar manner to assess the biological and performancecharacteristics of the compounds. Other detection methods include, butare not limited to flow cytometry, immunoassays, hybridization assays,and microarray analysis, can also be used in the practice of thisdisclosure.

(b) Therapeutic Applications

Certain of the imaging compounds as described herein, for example,imaging compounds containing a fluorochrome and/or a drug molecule, canbe used to ameliorate a symptom of, or treat, a particular healthcondition, such as a disease or disorder. The method comprises (a)administering to a subject an amount of one or more the imagingcompounds described herein sufficient to impart a therapeutic effect inthe subject; and (b) permitting sufficient time for the agent todistribute within the subject or otherwise localize in a region of thesubject to be treated and then, (c) depending upon the therapeuticagent, optionally activating the imaging compound to impart atherapeutic effect. For example, when the therapeutic agent is aradiolabel, no subsequent activation is required. However, when thetherapeutic agent is a photoreactive agent, for example, a dye used inphotodynamic therapy, the agent may be activated by exposing the agentto light having a wavelength that activates the agent. As a result, theagents can be used to treat a condition of interest, for example, acancer, immune disorder, inflammatory disorder, vascular disorder andthe like. Furthermore, the agents can be used to prevent, ameliorate, orreverse angiogenesis in a region of interest in the subject.

Various aspects of the present disclosure are illustrated by thefollowing non-limiting examples. The examples are for illustrativepurposes and are not a limitation on any practice of the presentinvention. It will be understood that variations and modifications canbe made without departing from the spirit and scope of the invention.

EXAMPLES Example 1: Synthesis of Fluorochrome Compound D65 (15 in Scheme1)

1-ethylbenzo[cd]indol-2(1H)-one (2)

2.5 g Benz[cd]indol-2(1H)-one (1, from TCI America) was dissolved in 20mL of dry DMF, to which was added 2 equivalents of sodium hydride (60%in mineral oil) after washing with dry hexanes under nitrogen cooled to0-5° C. with stirring. After 5 minutes, three equivalents of ethyliodide were added in drops over 2 min. Cooling was removed and wasallowed to warm up to room temp; stirring was continued for 2 hrs. Thereaction was monitored by TLC (Silica, Hexane-EtOAc 3:1) as well as byRP IPLC on C18 column (Gradient: 10%-85% B; B=Acetonitrile and A=25 mMAmmonium Formate with 5% methanol). Reaction mixture was poured into icewater, and the product was extracted with ethyl acetate followed bywashings, drying over anhydrous sodium sulfate, filtration and rotaryevaporation which afforded a yellow solid in 90% yield.

Sulfonyl Chloride of 1-ethylbenzo[cd]indol-2(1H)-one (3)

To 1-ethylbenzo[cd]indol-2(1H)-one (2, 2 g) in an RBF was added 20 mL oftrichloroethylene (TCE) and was fitted with a reflux condenser. Threeequivalents of chlorosulfonic acid (ClSO₃H) was added in drops over 2min with magnetic stirring. Reaction mixture was heated to 80° C. After16 hrs, the contents were concentrated on a rotovap and then poured intowater. Extraction with ethyl acetate and washings with water severaltimes, followed by rotovap drying yielded the sulfonyl chloride as theexclusive monosulfonyl chloride product, which was confirmed LCMS as thesulfonic acid MW 277 and by proton NMR (300 Mhz) as K salt of sulfonicacid in DMSO-d₆: δ (ppm) (JJ coupling constants in Hz): 1.254 (t, 3H),3.921 (q, 2H), 7.084 (d, 1H, JJ=7.44), 7.789 (t, 1J, JJ=7.14), 7.785 (d,1H, JJ=7.41), 8.024 (d, 1H, 7.14), 8.722 (d, 1H, JJ=8.52)

2,2,2-Trifluoro-1-p-tolyl-ethanol protection of sulfonyl chloride (6)(Ref.: Pauff, S. M., et. al., J. Org. Chem. 2013, 78, 711-716)1-ethylbenzo[cd]indol-2(1H)-one-6-sulfonylchloride (3) as obtained abovewas treated with 2,2,2-Trifluoro-1-p-tolyl-ethanol (at 5% excess) indichloromethane and two equivalents of triethylamine at RT, withstirring for 16 hrs. The product was monitored by TLC, and confirmed byLCMS. Aqueous extraction and washings with 0.1M NaOH followed by rotovapdrying the sulonate ester was isolated as thick oil.

Grignard Reaction to 7: The sulfonate ester was cooled to 0° C. in dryTHF under nitrogen. Two equivalents of 3.0 Molar THF solution of methylmagnesium chloride (MeMgCl) was added through a syringe and then allowedto warm up to room temp. After 2 hrs, 1M HCl was added, and rotovapdried to remove THF. The resulting mass was extracted into ethylacetate, washed with water and 7 was isolated as greenish blue solid.

The quaternary salt 7 obtained as above was immediately deprotected with4% aqueous FTA at room temp for 2 hrs, which was confirmed by LCMS. Theresulting deprotected quaternary salt was extracted into water, and theorganic materials were removed by washing with ethyl acetate. Thoroughdrying by rotovap and then speed vac overnight afforded the dryquaternary salt 8.

Preparation of Schiff's base (9) was carried out as described in theU.S. Pat. No. 9,798,604.

Preparation of diester 10 was carried out using 100 mg of the quaternarysalt 8 dissolved in 5 mL of acetic acid, 10 mL acetic anhydride in a 50mL RBF to which was added 0.5 equivalent of bisanil 9. Two equivalentsof sodium acetate were added and the reaction mixture was heated at 100°C. for 8 hrs. The reaction mixture was concentrated on rotovap, and theresidue was precipitated in ethyl acetate, which was then centrifuged ina 50 mL polypropylene tube, washed 1× with ethyl acetate, and then speedvac dried. The solid was dissolved in water and purified on C18 columnby HPLC (Gradient: 10-60% B; Mobile phases B=Acetonitrile and MobilePhase A=25 mM Triethyl ammonium acetate, pH 6.7 with 5% acetonitrile).Pure product was obtained in 25% yield of the dye reaction.

Preparation of 11: The diester 10 was dissolved in water and 5M NaOHsolution was added so that the final net conc. being 500 mM. After 2hrs, the saponification was completed as indicated by LCMS and the monoacid ester was purified by RP HPLC on C18 column (Gradient: 10-50% B;Mobile phases B=Acetonitrile and Mobile Phase A=25 mM Triethyl ammoniumacetate, pH 6.7 with 5% acetonitrile). The collected fractions frommultiple runs were combined and rotovap dired. The residue was dissolvedin minimal amount of DMF and precipitated with ethyl acetate,centrifuged and then the solid was speed vac dried at 30 C for 1 h.

Preparation of 12: To dry mono acid ester 11 in 1 mL of dry DMF wasadded 2 equivalents of HATU (Aldrich,N-[(Dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminiumhexafluorophosphate N-oxide, CAS #148893-10-1), three equivalents oftaurine, and three equivalents of DIPEA (N,N-di-isopropyl ethyl amine),and incubated at 37° C. for 1 h. After the completion of couplingreaction was revealed by LCMS, it was diluted into water and immediatelypurified on C18 column as above with a gradient of 05-40% B. Theresulting pure fractions were combined, rotovap dried followed by EtOAcprecipitation.

Preparation of 13: Compound 12 was treated with 0.25 M solution oflithium hydroxide at room temp for two hours. The saponification wasconfirmed by LCMS. Acetic acid was added to neutralize the the reactionmixture, and was purified on C18 column with a gradient of 05-30% B. Thepure fractions were combined and rotovap dried. Dry dark brown powderwas isolated by precipitation in DMF-ethyl acetate (1:20).

Preparation of 14: It was carried out in two steps. Compound 13 wasmixed with 2 equivalents of DSC (disuccinimidyl dicarbonate) in dry DMF(500 μL), and 2 equivalents of N-methyl morpholine (NMM) was added.Mixed and incubated at 37° C. for 1 h. The NHSE formation was confirmedby LCMS as butylamine product (a small aliquot (1 uL) of the reactionmix was added to 50 μL of 0.1M butylamine-HCl, pH 9, and analyzed byLCMS). The DMF solution was precipitated with ethyl acetate andcentrifuged to isolate the NHSE. It was immediately used as such in thenext reaction, by redissolving in dry DMF and reacting with fiveequivalents of 6-amino hexanoic acid, 2 equivalents of NMM andincubating at 37° C. for 1 h. The product formation and the completionof the reaction were confirmed by LCMS, after which the reaction mixturewas acidified with acetic acid and diluted with water followed bypurification on C18 column using a gradient of 05-30% B. The collectedfractions were dried by rotovap and the residue was precipitated by DMFand ethyl acetate (1:20). Centrifuging the precipitate and then dryingthe residue in speed vac at 30° C. for 30 minutes, afforded the darkpowder.

Preparation of 15: Compound 14 (5 mg) was mixed with 2 equivalents ofDSC (disuccinimidyl dicarbonate) in dry DMF (500 μL), and 2 equivalentsof N-methyl morpholine (NMM) was added. Mixed and incubated at 37° C.for 1 h. The NHSE formation was confirmed by LCMS as butylamine product(a small aliquot (1 uL) of the reaction mix was added to 50 μL of 0.1Mbutylamine-HCl, pH 9, and analyzed by LCMS). The DMF solution wasprecipitated with ethyl acetate and centrifuged to isolate the NHSE. Itis stored at −20° C. and can be used for labeling reactions.

Example 2: Synthesis of Fluorochrome Compound D66 (24 in Scheme 2)

1-ethylbenzo [cd] indol-2(1H)-one

2.5 g Benz[cd]indol-2(1H)-one (1, from TCI America) was dissolved in 20mL of dry DMF, to which was added 2 equivalents of sodium hydride (60%in mineral oil) after washing with dry hexanes under nitrogen cooled to0-5° C. with stirring. After 5 minutes, three equivalents of ethyliodide was added in drops over 2 min. Cooling was removed and wasallowed to warm up to room temp; stirring was continued for 2 hrs. Thereaction was monitored by TLC (Silica, Hexane-EtOAc 3:1) as well as byRP IPLC on C18 column (Gradient: 10%-85% B; B=Acetonitrile and A=25 mMAmmonium Formate with 5% methanol). Reaction mixture was poured into icewater, and the product was extracted with ethyl acetate followed bywashings, drying over anhydrous sodium sulfate, filtration and rotaryevaporation which afforded a yellow solid in 90% yield.

5,6-Di-sulfonyl Chloride of 1-ethylbenzo[cd]indol-2(1H)-one (16)

To 1-ethylbenzo[cd]indol-2(1H)-one (2) in an RBF was added 25 mL ofchloroform (CHCl₃) and was fitted with a reflux condenser. Fiveequivalents of chlorosulfonic acid (ClSO₃H) was added in drops over 2min with magnetic stirring. Reaction mixture was heated to reflux for 24hrs. The contents were concentrated on rotovap and then poured intowater and was washed with ethyl acetate. Aqueous layer on analysis byLCMS confirmed only one product whose molecular weight of 357corresponded to the di-sulfonic acid form of the product 16. It wasconverted to potassium salt by treating with 1.05 equivalent of KOH. Oneequivalent of 18-crown-6 was further added to the mixture and stirredwell, to make it soluble in organic solvents to be suitable for use inthe next step. The resulting mass was dried by rotovap thoroughly.

The positions of sulfonate substitutions in 17 to be ortho and para to Nwere confirmed by proton NMR (300 MHz) in DMSO-d6 (as K salt of sulfonicacid) δ (ppm) (JJ coupling constants in Hz): 1.225 (t, 3H), 4.489 (q,2H), 7.758 (t, 1H, JJ=8.2), 7.976 (d, 1H, JJ=6.9), 8.338 (s, 1H), 8.722(d, 1H, JJ=8.5)

Grignard Reaction to 18: The di-sulfonated benz[c,d]-indole-one-K-crownether complex was cooled to 0° C. in dry THF under nitrogen. Twoequivalents of 3.0 Molar THF solution of methyl magnesium chloride(MeMgCl) was added through a syringe and then allowed to warm up to roomtemp. After 2 hrs, 1M HCl was added, and rotovap dried to remove THF.The resulting mass was extracted into ethyl acetate, washed with waterand crude 18 isolated as bluish green solid was thoroughly dried byrotovap and speed vac.

Preparation of Schiff's base (9) was carried out as described in theU.S. Pat. No. 9,798,604.

Preparation of diester 19 was carried out in the same manner asdescribed for compound 10 in Scheme 1. Purification was carried out onRP-C18 column by HPLC (Gradient: 10-50% B; Mobile phases B=Acetonitrileand Mobile Phase A=25 mM Triethyl ammonium acetate, pH 6.7 with 5%acetonitrile).

Preparation of 20: The diester 19 was dissolved in water and 5M NaOHsolution was added so that the final net conc. being 500 mM. After 2hrs, the saponification was completed as indicated by LCMS and the monoacid ester was purified by RP HPLC on C18 column (Gradient: 5-40% B;Mobile phases B=Acetonitrile and Mobile Phase A=25 mM Triethyl ammoniumacetate, pH 6.7 with 5% acetonitrile). The collected fractions frommultiple runs were combined and rotovap dired. The residue was dissolvedin minimal amount of DMF and precipitated with ethyl acetate,centrifuged and then the solid was speed vac dried at 30 C for 1 h.

Preparation of 21: To dry mono acid ester 20 in 1 mL of dry DMF wasadded 2 equivalents of HATU (Aldrich,N-[(Dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminiumhexafluorophosphate N-oxide, CAS #148893-10-1), three equivalents oftaurine, and three equivalents of DIPEA (N,N-di-isopropyl ethyl amine),and incubated at 37° C. for 1 h. After the completion of couplingreaction was revealed by LCMS, it was diluted into water and immediatelypurified on C18 column as above with a gradient of 00-30% B. Theresulting pure fractions were combined, rotovap dried followed by EtOAcprecipitation.

Preparation of 22: Compound 21 was treated with 0.25 M solution oflithium hydroxide at room temp for two hours. The saponification wasconfirmed by LCMS. Acetic acid was added to neutralize the the reactionmixture, and was purified on C18 column with a gradient of 00-30% B. Thepure fractions were combined and rotovap dried. Dry dark brown powderwas isolated by precipitation in DMF-ethyl acetate (1:20).

Preparation of 23 was carried out in two steps. Compound 22 was mixedwith 2 equivalents of DSC (disuccinimidyl dicarbonate) in dry DMF (500μL), and 2 equivalents of N-methyl morpholine (NMM) was added. Mixed andincubated at 37° C. for 1 h. The NHSE formation was confirmed by LCMS asbutylamine product (a small aliquot (1 uL) of the reaction mix was addedto 50 μL of 0.1M butylamine-HCl, pH 9, and analyzed by LCMS). The DMFsolution was precipitated with ethyl acetate and centrifuged to isolatethe NHSE. It was immediately used as such in the next reaction, byredissolving in dry DMF and reacting with five equivalents of 6-aminohexanoic acid, 2 equivalents of NMM and incubating at 37° C. for 1 h.The product formation and the completion of the reaction were confirmedby LCMS, after which the reaction mixture was acidified with acetic acidand diluted with water followed by purification on C18 column using agradient of 00-30% B. The collected fractions were dried by rotovap andthe residue was precipitated by DMF and ethyl acetate (1:20).Centrifuging the precipitate and then drying the residue in speed vac at30° C. for 30 minutes, afforded the dark powder.

Preparation of 24: Compound 23 was mixed with 2 equivalents of DSC(disuccinimidyl dicarbonate) in dry DMF (500 μL), and 2 equivalents ofN-methyl morpholine (NMM) was added, and mixed well. It was incubated at37° C. for 1 h. The NHSE formation was confirmed by LCMS as butylamineproduct (a small aliquot ˜1 uL of the reaction mix was added to 50 μL ofa solution of 0.1M butylamine-HCl (pH˜9) and analyzed by LCMS). The DMFsolution was precipitated with ethyl acetate and centrifuged to isolatethe NHSE. It was stored at −20° C. and was used for labeling reactionslater.

Example 3: Synthesis of Fluorochrome Compound D67 (28 in Scheme 3)

Preparation of Compound 26: The same method used for synthesizingcompound 10 was followed and the synthesis confirmed by massspectroscopy as 687.13 Da.

Preparation of Compound 27: A mixture of 10 mg of chloro dye 26dissolved in 200 μL of anhydrous DMF, five equivalents of mercaptopropionic acid (MpA) and five equivalents of pyridine (dried over KOH)in a 2 mL tube was incubated at 37° C. for 1 hr. It was quenched with 25μL acetic acid, diluted in water and purified on C18 column (gradient10-65% B) to get 40% yield. The LCMS for 27 confirmed the MW of 756 Da.

Preparation of Compound 28: It was prepared following the same methodused for compound 15.

Chloro dye 26 exhibits a significantly different absorption property invarious solvents, with its abs.max ranging from 810 nm indichloromethane (DCM) to 1069 nm in DMSO as shown in Table 11 below.

The large shifting of abs. max to lower wavelengths, especially in wateris noteworthy because it is desirable that such “blue shifts” areminimal to be useful in biological applications such as in vivo imaging.Such shifts are common to cyanine dyes and are attributable toaggregation or stacking of the dye molecules with each other. The higherdegree of aggregation is reflected in the larger “nm” shifts towardsblue spectrum.

TABLE 11 Absorbance maxima of 26 in various solvents No SolventAbsorption max (nm) 1 Methanol 1022 2 Ethanol 1032 3 DMSO 1065 4Dichloromethane 810 5 Chloroform 820 6 Acetone 833, 1033 7 Acetonitrile1019 8 Water 829

Example 4: Synthesis of Fluorochrome Compound D68 (34 in Scheme 4)

The compounds 30 to 34 were prepared in a similar manner as that ofcompounds 26 to 28 described in Scheme 3.

Compound 18 (Potassium disulfo-1-ethyl-2-methyl benz [c,d]-indolinineinner salt) is used in place of 8 in Schemes 3 and 4 for the synthesisof tetra sulfonated analogs.

Example 5: Synthesis of Fluorochrome Compound D29 (39 in Scheme 5)

Compound 36

To 0.5 mmol of commercially available sodium(4-acetyl-benzene)sulfonatein a pressure tube was added 1 mL of 70% perchloric acid and 0.2 mL ofethyl acetate. The air tightly closed tube was heated on an oil bath at120° C. for 16 hrs. A large amount of cold acetone (50 mL) was added tothe cooled tube, and the precipitated product was collected and dried.LCMS confirmed the product of MW 407 Da, and was purified on PhenyHexyl(X-Bridge) column with a gradient of 5-40% B. A 25% yield was realized.

Compound 37

0.5 mmol of compound 36 was dissolved in a pressure tube containing 5 mLof a solution prepared as below: 2 g of sodium sulfide (as nano hydrate)in 5 mL water and 2 mL ethanol to which was added 0.75 g of sodiumbicarbonate (NaHCO₃) was added and the tube was quickly screw capped andleft stirring at room temp for 12 hrs. The contents were concentrated byrotovap and the residue was dissolved in water and purified on RPC18column (10-65% B).

Compounds 38 and 39

They were prepared by following the same procedure described forcompounds 26 and 27 respectively.

Example 6: Synthesis of Fluorochrome Compounds D37 and D38 (Compounds 50and 51 in Scheme 6)

Compound 42

Compound 40 (TCI America), 1.08 g was mixed with 5 mL of acetone and 15mL of methanol in an RBF. 1.04 mL of 50% sodium hydroxide was dilutedwith 1.6 mL of water and then added to the flask with vigorous stirring.With in 10 min, 90% product was realized by HPLC analysis (00-50% B,PhenyHexyl column, 25 mM ammonium formate and acetonitrile). It waspurified after quenching with dil HCl and concentrating on rotovap. 60%yield. In the same manner compound 43 can be synthesized fromcommercially available 41.

Compound 44

0.17 g of compound 42 was dissolved in 1 mL of 25 mM triethylammoniumacetate buffer (TEAAc, pH4.7) in a 50 mL poly propylene tube, to whichwas added 4.1 mL of acetonitrile and 0.35 mL of thiophenol. Thehomogeneous solution was incubated at 37° C. for 16 hrs. The reactionmixture was diluted with water and extracted with dichloromethane toremove thiophenol. The aqueous layer was subjected to purification byRP-HPLC on Phenylhexyl column, using 25 mM TEAAc (A) at pH4.7 andacetonitrile (B) as mobile phases with a gradient of 00-50% B. The purefractions were collected and dried on speed vac. LCMS confirmed the massof 337. In the same manner compound 45 can be synthesized from 43.

Compound 46

To 16 mg of dried compound 44 in a 2 mL glass vial, was added 0.15 mL oftrifluoromethane sulfonic acid inside a fume hood. The capped vial wasleft at 37° C. for 12 hrs. The reaction mixture was dilated with waterwhich formed pink solution leaving insoluble gummy precipitate. Aqeuouslayer was separated by centrifuging and subjected to HPLC purificationand the peak that corresponded to mass 317 Da was isolated (20% yield).Compound 47 can be synthesized from 45 in the same manner.

Compound 48

Prepared by following the procedure described for the preparation ofcompound 26. Compound 49 can be prepared from compound 47 in the sameway.

Compound 50 was prepared by following the procedure described for thepreparation of compound 27.

Compound 51 can be made in a similar manner.

Example 7: Synthesis of Fluorochrome Compounds D69 and D70 (64 and 65 inScheme 7)

Compound 52

Ten mmoles of 2-formylbenzenesulfonic acid (as sodium salt, TCI) ismixed with five mmoles of acetone in 96% ethanol (20 mL) and Ba(OH)₂ (2mmoles) is added as catalyst, and the mixture is refluxed for 2 hrs(Ref. Sinistierra, J. V., et al., Synthesis (6), 502-4 (1984)). Thereaction mixture is neutralized with dilute HCl, and is purified byRP-HPLC on C18 column (gradient 05-50% B, A=95% 25 mM TEAAc+5%acetonitrile, pH 6.7, B=100% acetonitrile). The product is identified byLCMS corresponding to MW 394 Da.

Compound 54

Compound 54 is prepared by following the procedure described inWO2016/081813 on page 41 (Detty, et.al.), and the product is purified byRP-HPLC on C18 column with a gradient of 10-60% B. Compound 54 is thenconverted to potassium salt by mixing with 1.05 equivalent of 1 M KOHfollowed by adding 1.05 equivalent of 18-Crown-6. It is concentrated byrotovap and then dried under vacuum for an over night at 37° C.resulting in compound 56.

Compound 58

Compound 56 is oxidized to 58 by DDQ in toluene and refluxing for 2 hrs(Detty, et.al., WO2016/081813, page 42), followed by filtration throughcelite and rotovap drying.

The resulting 58 is reacted with a THF solution of 3.0 M MeMgCl in THFunder nitrogen at room temp, followed by quenching with an aqueoussolution of 10% HPF6. THF and water are removed by rotovap. The residueis redissolved in ethyl acetate, and washed gently with water to removeinorganic salts. Rotovap drying afford the crude 4-methyl pyrylium salt60 which is used in the synthesis of dyes as such.

Compound 62 is synthesized by the procedure described for compound 10.

Compound 64 is made as per the procedure for compound 27.

Compound 65: The synthesis of compound 65 from compound 41 (4-formyl,1,3 benzene sulfonic acid, di sodium slat, TCI) is carried out in thesame way as described above for compound 64.

Example 8: Synthesis of Fluorochrome Compound D23 (76 in Scheme 8)

Compound 67

Trimethyl silyl acetylene (66) is cooled at −78° C. in THF undernitrogen, to which is added ethyl formate slowly with stirring. After 10min, it is warmed up to room temp and stirred for an additional 2 hrs.Reaction is quenched with saturated ammonium chloride solution, and THFis removed by rotovap. The aqueous solution is extracted with ethylacetate, concentrated and dried by rotovap (Ref.: Bowling, N. P. et.al,J. Org Chem., 71, 5841-5847 (2006)).

Compound 68: Compound 67 is treated with Ba manganate in methylenechloride and isolated as described in the above ref. (Bowling, N. P.et.al, J. Org Chem., 71, 5841-5847 (2006)).

Compound 70 is prepared from 4-iodobenzenesulfonylchloride in the sameway as described for compound 6 previously.

Compound 71: The procedure reported in ref. Hatanaka, Y., and Hiyama,T., J. Org Chem., (1988), 53, 920-923 is followed for making compound 71from 68 and 70.

Compound 72: Compound 71 is treated with a solution of sodium sulfide inethanol water along with sodium hydroxide as described in ref.JP2001-011070A.

Compound 73: Compound 72 is subjected Grignard's reaction with MeMgCl inTHF and quenched with dil HPF6 solution, by following the proceduredescribe earlier for compound 60.

Compound 74: Deprotection of compound 73 is carried out in 96% TFA-4%water in the same way as done for the compound 8.

Compound 75: Synthesis of compound 10 procedure was followed using 74and bisanil 25.

Compound 76: Chloro dye 75 is converted to a vinyl thio ether 76 in DMFwith pyridine as catalyst as per the procedure previously described forcompound 27.

Example 9: Synthesis of Fluorochrome Compound D71 78 in Scheme 9)

Synthesis of compound 78 was carried out using 2 equivalents of compound8 (10 mg) and compound 77 in a mixture of 2.5 mL acetic acid, 5 mLacetic anhydride and 2 equivalents of sodium acetate and heating at 120°C. for 3 hrs. After removing the solvents by rotovap the residue wasdissolved in water and purified by RP-HPLC (using C18 column and agradient of 10-60% B (Mobile phase A=95% 25 mM TEAAc+5% acetonitrile, pH6.7, mobile phase B=100% acetonitrile). The product was identified byLCMS corresponding to MW 709 Da (as M+1). The absorption max of compound78 in methanol is determined to be 851 nm.

Example 10: Synthesis of Fluorochrome Compound D72

To the mixture of the quaternary salt (42 mg, 145 umol), the bridge (25mg, 65 umol), cesium acetate (150 mg, 780 umol) was added a mixture ofacetic anhydride and acetic acid (2:1, v/v, 3 mL). The mixture wasstirred at ˜20° C. for 4 hr. The mixture was concentrated and theproduct was purified by solid phase extraction on a packed C-18 columnusing aqueous trimethylamine acetate buffer and acetonitrile as theeluent. Mw calc: 740.2, found: 741.3. Abs λmax: 1026 (methanol). Thechloro-bicyclo bridge was synthesized as per the procedure described inU.S. Pat. No. 8,221,721.

Example 10: Synthesis of Fluorochrome Compound D73

To the mixture of the quaternary salt (18 mg, 69 μmol), the bridge (12mg, 34 μmol), cesium acetate (120 mg, 625 μmol) was added a mixture ofacetic anhydride and acetic acid (2:1, v/v, 3 mL). The mixture wasstirred at ˜20° C. for 16 hr. The mixture was concentrated and theproduct was purified by solid phase extraction on a packed C-18 columnusing aqueous trimethylamine acetate buffer and acetonitrile as theeluent. Mw calc: 684.1 Da, found: 684.2 Da. Abs λmax: 1050 nm(methanol).

Example 11: Synthesis of Fluorochrome Compound D74

To the mixture of the quaternary salt (15 mg, 45 μmol), the bridge (TCI,6 mg, 21 μmol), cesium acetate (90 mg, 470 μmol) was added a mixture ofacetic anhydride and acetic acid (2:1, v/v, 3 mL). The mixture wasstirred at ˜20° C. for 5 hr. The mixture was concentrated and theproduct was purified by solid phase extraction on a packed C-18 columnusing aqueous trimethylamine acetate buffer and acetonitrile as theeluent. mw calc: 728.2 Da, found: 729.2 Da. Abs λmax: 980 nm (methanol).

Example 12: Synthesis of Fluorochrome Compound D19

To the mixture of the quaternary salt (31 mg, 100 μmol), the bridge(11.4 mg, 40 μmol), cesium acetate (202 mg, 1.0 mmol) was added amixture of acetic anhydride and acetic acid (2:1, v/v, 3 mL). Themixture was stirred at ˜40° C. for 4 hr. The mixture was concentratedand the product was purified by solid phase extraction on a packed C-18column using aqueous trimethylamine acetate buffer and acetonitrile asthe eluent. mw calc: 672.0 Da, found: 671.1 Da. Abs λmax: 980 nm(methanol).

Example 13: Synthesis of Fluorochrome Compound D18

To the mixture of the disulfonate quaternary salt (17 mg, 42 μmol), thesulfonate acetic acid quaternary salt (13 mg, 42 μmol), the bridge (10mg, 35 μmol), cesium acetate (150 mg, 781 μmol) was added a mixture ofacetic anhydride and acetic acid (2:1, v/v, 3 mL). The mixture wasstirred at ˜20° C. for 18 hr. The mixture was concentrated and theproduct was purified by preparative HPLC using aqueous trimethylamineacetate buffer and acetonitrile as the eluent. mw calc: 736.8 Da, found:737.7 Da. Abs λmax: 970 nm (methanol).

Example 14: Synthesis of Fluorochrome Compound D75

To the mixture of the quaternary salt (25 mg, 61 umol), the bridge (13mg, 28 umol), cesium acetate (120 mg, 625 umol) was added a mixture ofacetic anhydride and acetic acid (2:1, v/v, 3 mL). The mixture wasstirred at ˜40° C. for 4 hr. The mixture was concentrated and theproduct was purified by solid phase extraction on a packed C-18 columnusing aqueous trimethylamine acetate buffer and acetonitrile as theeluent. mw calc: 984.2, found: 985.1. Abs λmax: 990 nm (methanol)

Example 15: Synthesis of 4-methyl-2,6-bis(3-sulfophenyl)thiopyryliumchloride

2,6-Diphenyl-4H-thiopyran-4-one (0.5 g) The thiopyranone was dissolvedin 5 mL of chloroform and stirred in a 250 mL round bottom flask.Chlorosulfonic acid (1.25 mL) was then added slowly. A reflux condenserwas attached and the solution was heated to 70° C. overnight. Thereaction mixture was then poured over ice and the solid precipitateisolated by filtration, washed with ice water and dried under vacuum togive 2,6-di(chlorosulfophenyl)-4H-thiopyran-4-one. ESI MS calculated[M+H]=460.9 for C₁₇H₁₁C₁₂O₅S₃ ⁺, found 461.1.

2,6-di(chlorosulfophenyl)-4H-thiopyran-4-one (200 mg), potassiumcarbonate (120 mg) and 18-crown-6 (230 mg) were combined in 2 mL ofwater and 1 mL of acetonitrile and heated to 60° C. for 15 minutes. Thesolution was diluted to 5 mL with water and passed through a 2 g columnof C18 reverse phase silica gel eluting the product with 20%acetonitrile in water. The first 5 mL of eluent were combined and driedunder vacuum to give 405 mg of 2,6-di(sulfophenyl)-4H-thiopyran-4-one,potassium/18-crown-6 salt. ESI MS calculated [M+H]=425.0 for C₁₇H₁₃O₇S₃⁺, found 425.2.

2,6-di(sulfophenyl)-4H-thiopyran-4-one, potassium/18-crown-6 salt (480mg) was dispersed in 5 mL of anhydrous THF in a 250 mL round bottomflask flushed with nitrogen. Methylmagnesium chloride (0.94 mL of a 3 Msolution in THF) was added dropwise, and the mixture stirred at roomtemperature overnight. The solution was then cooled in an ice bath andquenched by slow addition of 4 mL of 1 M hydrogen chloride in diethylether. The resulting yellow, solid precipitate of4-methyl-2,6-bis(3-sulfophenyl)thiopyrylium chloride was filtered,washed with ether and dried in vacuum. ESI MS calculated [M+H]=423.0 forC₁₈H₁₅O₆S₃ ⁺, found 423.2.

Example 16: Synthesis of Fluorochrome Compound D60

4-methyl-2,6-bis(3-sulfophenyl)thiopyrylium chloride (12 mg), andN-[(3-(Anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]anilinemonohydrochloride (5 mg) were combined in 0.5 mL of acetic anhydride and0.5 mL of acetic acid. Triethylamine (TEA) (0.1 mL) was added and themixture was heated to 95° C. for 1.5 h. The crude product wasprecipitated with 25 mL of diethyl ether, filtered and purified by HPLCto give dye D60. ESI MS calculated [M+H]=981.0 for C₄₄H₃₄ClO₁₂S₆ ⁺,found 981.5. Absorbance max (MeOH) 1062 nm.

Example 17: Synthesis of Fluorochrome Compound D64

4-methyl-2,6-bis(3-sulfophenyl)thiopyrylium chloride (12 mg), and2-chlorocyclopent-1-ene-1,3-dialdehyde (2.2 mg) were combined in 0.5 mLof acetic anhydride and 0.5 mL of acetic acid. Triethylamine (TEA) (0.1mL) was added and the solution was stirred at room temperature for 4 h.The crude product was precipitated with 25 mL of diethyl ether, filteredand purified by HPLC to give dye D64. ESI MS calculated [M+H]=967.0 forC₄₃H₃₂ClO₁₂S₆ ⁺, found 967.4. Absorbance max (MeOH) 1068 nm.

Example 18: Synthesis of Fluorochrome Compound D61

4-methyl-2,6-bis(3-sulfophenyl)thiopyrylium chloride (12 mg), andN-3-chloro-4-((phenylamino)methylene)bicyclo[3.2.1]oct-2-en-2-yl)methylene)benzenaminiumchloride (5.4 mg) were combined in 0.5 mL of acetic anhydride and 0.5 mLof acetic acid. Triethylamine (TEA) (0.1 mL) was added and the mixturewas heated to 95° C. for 1.5 h. The crude product was precipitated with25 mL of diethyl ether, filtered and purified by HPLC to give compoundD61. ESI MS calculated [M+H]=1007.0 Da for C₄₆H₃₆ClO₁₂S₆ ⁺, found 1007.4Da. Absorbance max (MeOH) 1063 nm.

Example 19. Synthesis of Fluorochrome Compound D62

The chloro-substituted thiopyrylium dye D64 (14 mg) and4-mercaptophenylacetic acid (4.4 mg) were combined and dissolved in 0.5mL of anhydrous DMF to which 0.01 mL of pyridine was added. The mixturewas stirred at room temperature for 30 minutes, then the productcompound D56 was precipitated with ether, isolated by filtration andpurified by HPLC. ESI MS calculated [M+H]=1099.0 Da for C₅₁H₃₉O₁₄S₇ ⁺,found 1099.4 Da. Absorbance max (MeOH) 1061 nm.

Example 20: Synthesis of Fluorochrome Compound D63

4-methyl-2,6-bis(3-sulfophenyl)thiopyrylium chloride (3 mg), andN-5,5-bis(ethoxycarbonyl)-3-((phenylamino)methylene)cyclohex-1-en-1-yl)methylene)benzenaminiumchloride (1.7 mg) were combined in 0.5 mL of acetic anhydride and 0.5 mLof acetic acid. Triethylamine (TEA) (0.1 mL) was added and the mixturewas heated to 95° C. for 1.5 h. The crude product was precipitated with25 mL of diethyl ether, filtered and purified by HPLC to give dye D63.ESI MS calculated [M+H]=1091.1 Da for C₅₀H₄₃O₁₆S₆ ⁺, found 1091.6 Da.Absorbance max (MeOH) 1016 nm.

Example 21: Synthesis of 9,10-dimethyl-3,6-disulfoacridin-10-ium

9-methylacridine (0.5 g) was placed in a 250 mL flask and immersed in anice bath. 1.2 mL of chlorosulfonic acid (1.2 mL) was added dropwise andthe mixture was stirred to obtain a clear, orange-brown solution. Areflux condenser was attached and the flask was heated to 100° C. for 12h. The mixture was then cooled in an ice bath and quenched by additionof 25 g of crushed ice. The mixture was filtered, and the solidprecipitate washed on the filter with 25 mL of ice water. The solid wascollected and transferred to a flask with 25 mL of 200 mM sodiumcarbonate, then heated to 70° C. with stirring for 1 h forming a clear,brown solution. The water was removed by evaporation, and the solidpurified by HPLC eluting with 25 mM triethylammonium acetate andacetonitrile which was evaporated to yield 0.9 g of the triethylammoniumsalt of 9-methyl-3,6-disulfoacridine. ESI MS calculated [M+H]=354.1 Dafor C₁₄H₁₂NO₆S₂ ⁺, found 354.3 Da.

9-methyl-3,6-disulfoacridine triethylammonium salt (0.5 g) andiodomethane (800 mg) are dissolved in 10 mL of anhydrous DMF in apressure vessel. The vessel is sealed tightly and heated to 60° C. for 8h. The product is precipitated by addition of 25 mL of ethyl acetate,filtered and washed with 25 additional mL to yield9,10-dimethyl-3,6-disulfoacridin-10-ium as a triethylammonium salt.

Example 22: Synthesis of 9,10-dimethyl-2,7-disulfoacridin-10-ium

10-methylacridin-9-one (1.0 g) was dispersed in 10 mL of anhydrouschloroform in a 100 mL round bottom flask and cooled in an ice bath.Chloropsulfonic acid (1.9 mL) was added dropwise. After the addition,the flask was fitted with a condenser and heated to reflux for 6 h. Thereaction was quenched by addition of 25 g of crushed ice and solidproduct isolated by filtration, washed with three portions of ice waterand dried in vacuum to give 10-methylacridin-9-one-2,7-disulfonylchloride.

10-methylacridin-9-one-2,7-disulfonyl chloride (0.5 g) was dissolved in10 mL of dichloromethane in a 100 mL round bottom flask and cooled in anice bath. 2,2,2-trifluoro-1-(p-tolyl)ethanol (0.58 g) dissolved in 3 mLof dichloromethane was then added followed by 0.52 mL of anhydroustriethylamine was added and the mixture was stirred for 10 minutes thenallowed to warm to room temperature. The solution was stirred for 3 h,then 15 mL of saturated sodium bicarbonate and the product was extractedwith three 25 mL portions of dichloromethane. The dichloromethaneextracts were combined, washed with saturated brine, dried over sodiumsulfate and evaporated to dryness to yield 0.553 g of2,2,2-trifluoro-1-(p-tolyl)ethanol protected10-methylacridin-9-one-2,7-disulfonate. ESI MS calculated [M+H]=714.1 Dafor C₃₂H₂₆F₆NO₇S₂ ⁺, found 714.2 Da.

2,2,2-trifluoro-1-(p-tolyl)ethanol protected10-methylacridin-9-one-2,7-disulfonate (0.4 g) was placed in anoven-dried 3-neck round bottom flask flushed with nitrogen. 10 mL ofanhydrous THF was added to the flask and mixture was stirred.Methylmagnesium chloride, 3 M in THE was added dropwise. The clear, redsolution was stirred at RT for 30 minutes, then cooled in an ice bath.2.4 mL of 1 M HCl was added dropwise then the solution was allowed towarm to room temperature. The THF was removed by rotary evaporation and25 mL of saturated sodium bicarbonate added to the solid residue thatwas then extracted into three portions of 25 mL of dichloromethane. Thedichloromethane extracts were washed with brine, dried over sodiumsulfate and the solvent removed under vacuum to give 0.39 g of2,2,2-trifluoro-1-(p-tolyl)ethanol protected9,10-dimethylacridinium-2,7-disulfonate. ESI MS calculated [M+H]=712.1Da for C₃₃H₂₈F₆NO₆S₂ ⁺, found 712.3 Da.

2,2,2-trifluoro-1-(p-tolyl)ethanol protected9,10-dimethylacridinium-2,7-disulfonate (0.39 g) was dissolved in 2.5 mLof trifluoroacetic acid (TFA) and 25 μL of water. The solution wasstirred at room temperature for 2 h. The deprotected product wasisolated by precipitation with ether to give the9,10-dimethyl-2,7-disulfoacridin-10-ium inner salt.

Example 23: Synthesis of Fluorochrome Compound D56

9,10-dimethyl-3,6-disulfoacridin-10-ium, triethylammonium salt (25 mg),andN-[(3-(Anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]anilinemonohydrochloride (10 mg) are combined in 0.5 mL of acetic anhydride and0.2 mL of acetic acid. Triethylamine (TEA) (0.02 mL) is added and themixture is heated to 80° C. for 8 h. The crude product is precipitatedwith 5 mL of ethyl acetate, filtered and purified by HPLC to give thechloro-substituted acridinium dye.

The chloro-substituted acridinium dye (15 mg) and 4-mercaptophenylaceticacid (5 mg) are combined and dissolved in 0.5 mL of anhydrous DMF towhich 0.01 mL of Pyridine is added. The mixture is stirred at roomtemperature for 30 minutes, then the product dye D56 is precipitatedwith ether, isolated by filtration and purified by HPLC.

Example 24: Synthesis of Fluorochrome Compound D59

9,10-dimethyl-2,7-disulfoacridin-10-ium inner salt (25 mg), and2-chlorocyclopent-1-ene-1,3-dialdehyde (5 mg) are combined in 0.5 mL ofacetic anhydride and 0.2 mL of acetic acid. Triethylamine (TEA) (0.02mL) is added and the mixture is heated to 50° C. for 4 h. The crudeproduct is precipitated with 5 mL of ethyl acetate, filtered andpurified by HPLC to give the chloro-substituted acridinium dye.

The chloro-substituted acridinium dye (15 mg) and 3-mercaptopropionicacid (5 mg) are combined and dissolved in 0.5 mL of anhydrous DMF towhich 0.01 mL of triethylamine (TEA) is added. The mixture is heated to60° C. for 2 h, then the product dye D59 is precipitated with ether,isolated by filtration and purified by HPLC.

Example 25: The Synthesis of 2H-naphtho[1,8-bc]thiophen-2-one

The only known cyanine dyes based on napthothiophenium salts found inthe literature were disclosed by Vasilenko, N. P.; Mikhailenko, F. A.;Maidannik, A. G. Khimiya Geterotsiklicheskikh Soedinenii (1987), (3),418-19 as the following:

However, such dyes are water insoluble and unsuitable for use inbiological applications. Hence, the following procedure describes thesynthesis of water soluble analogs of napthothiphenium derived symmetriccyanine dyes.

The synthesis of 2H-naphtho[1,8-bc]thiophen-2-one is carried outstarting from commercially available thionaphthol and trichloroaceticanhydride, substantially as described by Mitsuduo et.al in Synlett, 27(16), 2327-32 (2016). The resulting thio lactone is treated with 5-6fold excess of chlorosulfonic acid in chloroform for 24 hrs andhydrolyzed the sulfonyl chloride in water formingdisulfonated-2H-naphtho[1,8-bc]thiophen-2-one. It is converted tobis-tetrabutyl ammonium salt by treating with tetrabutyl ammoniumhydroxide. Grignard reaction is carried out with 3 molar THF solution ofMeMgCl under nitrogen in dry THF followed by acidic workup with HCl indiethylether yielding 2-methyl-6,8-disulfonaphtho[1,8-bc]thiophen-1-iumchloride, which is isolated by filtration and thorough drying undervacuum. It is reacted with bridge 9 in a mixture of acetic acid andacetic anhydride along with sodium acetate at 100° C. for about 8 hrs torealize the formation of tetra sulfonated napthothiophenium dye. It issubsequently subjected to a series of reactions as shown in the schemebelow to obtain the final product T8-8.

Scheme for the Synthesis of Dyes Based on P18

Example 26: Synthesis of Quaternary Salt Intermediates QS1 to QS6 Usedfor the Synthesis of Dyes with Endgroups P50, P32 and P51 to P54,Respectively

Several quaternary salts isolated by addition of HCl in ether arehygroscopic and decompose when exposed to moisture. In addition to thedesired quaternary salt, the isolated solid also contained other salts,i.e., potassium 18-crown-6 salt and magnesium salt from the Grignardreagent. Thus, the weight 00 purities were estimated based on the amountof starting amide used in the reaction and the materials were usedwithout further purification. Several exemplary quaternary saltintermediates are shown in Table 12.

TABLE 12 Quaternary Salts QS1 to QS6 used for the sysnthesis of dyesD81, D82, D72, D84, D85, D17, D19, D15, D13, D86, D20, D18 QuaternarySalts P Structure QS1

QS2

QS3

QS4

QS5

QS6

Synthesis of Quaternary Salt QS1

Alkylation. To the solution of benz[c,d] indol-2(1H) one (338 mg, 2.0mmol) and iodomethane (424 mg, 3.0 mmol) in dry DMF (5 mL) was addedpotassium t-butoxide (404 mg, 3.6 mmol) in portions over 10 min. After 1h at ambient temperature, the reaction was quenched by addition of 0.2mL of acetic acid. The solution was concentrated to ˜2 mL, andpartitioned with ethyl acetate (15 mL) and water (10 mL). The ethylacetate solution was washed with 2×10 mL of water and 10 mL of brine.After dried over anhydrous sodium sulfate, the solution was concentratedto dryness.

Weight: 330 mg, 91%. The structure was confirmed by LC/MS, m/z=184.4 Da,M+1.

Sulfonation. To starting material (184 mg, 1.0 mmol) was addedchlorosulfonic acid (500 μL) and the mixture was stirred at ambienttemperature for 16 h. The solution was carefully diluted with 2 mL ofwater and then quenched into a mixture of water/acetonitrile (2:1, v/v,30 mL) containing 6.0 g of triethylamine. The mixture was stirred atambient temperature for 16 h. The solution was concentrated to ˜10 mLand pH was adjusted to 4-5 with acetic acid. The mixture was loaded ontoa C18 packed column, 10 g resin and eluted with water. The column waseluted with a gradient mixture of aqueous buffer of triethylamine (12.5mM) and acetic acid (25 mM), TEAA buffer, and acetonitrile. The volumewas 20 mL each of the following TEAA/MeCN ratio (v/v): 95/5, 90/10,85/15, 80/20, and 75/25.

The yellow solution was collected and analyzed by LC/MS, m/z=263.5 Da.Concentration of the solution provided the product as a yellow solid astriethylamine salt with obtained weight: 296 mg, 81%.

Crown ether salt formation. To the solution of the triethylamine salt(183 mg, 0.50 mmol) in 5 mL of water was added potassium bicarbonate (50mg, 0.50 mmol) and 18-crown-6 (132 mg, 0.50 mmol), and the solution wasconcentrated to dryness. The solid was sonicated in 5 mL of MTBE and afine slurry resulted. The supernatant was removed after centrifugation.The solid was dried under vacuum to constant weight. Obtained weight ofcompound: 280 mg.

Grignard reaction. To the suspension of the crown ether salt (164 mg,0.29 mmol) in dry THE (5 mL) was added CH₃—MgCl (3 M in THF, 165 μL, 0.5mmol) and the resulting mixture was stirred at ambient temperature for 6h. To the mixture was added 2 mL of 1 M HCl in ether. The mixture wasstirred at ambient temperature for 30 min and collected bycentrifugation. The obtained solid was washed with 5 mL of ether and 2×2mL of 1 M aqueous HCl, and dried under vacuum. Obtained weight: 58 mg,76%. The structure was confirmed by LC/MS m/z=262.1 Da, M+1.

Synthesis of Quaternary Salt QS2

Alkylation. To the solution of benz[c,d] indol-2(1H)one (169 mg, 1.0mmol) and 1,3-propanesultone (183 mg, 1.5 mmol) in dry DMF (3.5 mL) wasadded potassium t-butoxide (202 mg, 1.8 mmol) in portions over 10 min.After 2 h at ambient temperature, the reaction was quenched by additionof 0.1 mL of acetic acid. The solution was concentrated to ˜1 mL byrotavap and diluted to 10 mL with water. The solution was loaded onto aC18 resin short column of 10 mL bed volume. The column was eluted with agradient mixture of aqueous TEAA and acetonitrile. The volume was 20 mLeach of the following TEAA/MeCN ratio (v/v): 95/5, 90/10, 85/15, 80/20,and 75/25. The yellow fractions were analyzed by LC/MS, m/z=292.4 Da,M+1. The appropriate fractions were combined and concentrated to drynessto a final obtained weight: 342 mg as the triethylamine salt, 87%. Thesolid was dissolved in 5 mL of water and solid potassium bicarbonate (88mg, 0.88 mmol) was added. The solution was concentrated to dryness toprovide the product as a yellow solid. The final obtained weight: 328mg.

Crown ether salt formation. To the solution of the potassium salt (66mg, 0.20 mmol) in 5 mL of water was added 18-crown-6 (53 mg, 0.20 mmol)and the solution was concentrated to dryness. The solid was sonicated in5 mL of MTBE and a fine slurry was resulted. The supernatant was removedafter centrifugation. The solid was dried under vacuum to constantweight. The obtained weight: 117 mg.

Grignard reaction. To the suspension of the crown ether salt (60 mg,0.10 mmol) in dry THE (3 mL) was added CH₃MgCl (3 M in THF, 60 μL, 0.18mmol) and the resulting mixture was stirred at ambient temperature for 4h. To the mixture was added a mixture of 1 M aqueous HCl (3 mL) andmethanol (1 mL). The mixture was stirred at ambient temperature for 30min and collected by centrifugation. The solid was washed with 2×5 mL ofdry ether and dried under vacuum. The obtained weight: 21 mg, 72%. Thestructure was confirmed by LC/MS, m/z=290.5 Da, M+1.

Synthesis of Quaternary Salt QS3

Sulfonation. To starting material (102 mg, 0.31 mmol) was addedchlorosulfonic acid (360 μL) and the mixture was stirred at ambienttemperature for 2 h. The solution was carefully diluted with 2 mL ofwater and then quenched into a mixture of water/acetonitrile (2:1, v/v,30 mL) containing 1.0 g of potassium carbonate. The mixture was stirredat ambient temperature for 16 h. The solution was concentrated to ˜10 mLand pH was adjusted to 4-5 with acetic acid. The mixture was loaded ontoa C18 packed column, 10 g resin and eluted with water. Two yellow bandsdeveloped and the first one was collected, while the second one wasretained in the column. The first yellow fraction was analyzed by LC/MS,m/z=372.5 Da, M+1. Concentration of the solution provided the product asa yellow solid, 108 mg, 78%.

Crown ether salt formation. To the solution of the di-potassium salt (50mg, 0.11 mmol) in 5 mL of water was added 18-crown-6 (58 mg, 0.22 mmol)and the solution was concentrated to dryness. The solid was sonicated in5 mL of MTBE and a fine slurry was resulted. The supernatant was removedafter centrifugation. The solid was dried under vacuum to an obtainedweight: 107 mg.

Grignard reaction. To the suspension of the crown ether salt (107 mg,0.11 mmol) in dry THE (3 mL) was added CH₃MgCl (3 M in THF, 60 μL, 0.18mmol) and the resulting mixture was stirred at ambient temperature for 4h. To the mixture was added HCl in ether (2 M, 300 μL) and ether (3 mL).The solid was washed with 2×5 mL of dry ether and dried under vacuum.The obtained weight: 167 mg, 27 w % based on 0.11 mmol of startingmaterial. Product not stable when analyzed by RP-HPLC and was usedwithout further purification.

Synthesis of Quaternary Salt QS4

Alkylation. To the solution of benz[c,d] indol-2(1H)one (250 mg, 1.48mmol) and methyl 4-iodobutylate (404 mg, 1.8 mmol) in dry DMF (5 mL) wasadded potassium t-butoxide (232 mg, 2.1 mmol) in portions over 10 min.After 2 h at ambient temperature, the reaction analyzed by TLC (Silica,1:1 ethyl acetate/hexane). A small amount of stating material wasdetected. The reaction was quenched by addition of 0.1 mL of aceticacid. The solution was concentrated to ˜2 mL by rotavap and diluted to10 mL with aqueous buffer of triethylamine (12.5 mM) and acetic acid (25mM). The solution was loaded onto a C18 resin short column of 10 mL bedvolume. The column was eluted with a gradient mixture of aqueous TEAAbuffer and acetonitrile. The volume was 10 mL each of the followingTEAA/MeCN ratio (v/v): 90/10, 80/20, 70/30, 60/40,50/50, and 60/40. Twoyellow fractions were collected and the second one was identified as thedesired product, m/z=270.3 Da by LC/MS. Concentration of the secondyellow fraction afforded the product. Final obtained weight: 291 mg,73%.

Sulfonation/Crown ether Salt. To starting material (200 mg, 0.74 mmol)was added chlorosulfonic acid (500 μL) and the mixture was stirred atambient temperature for 2 h. The solution was carefully diluted with 2mL of water and then quenched into a mixture of water/acetonitrile (2:1,v/v, 30 mL) containing 6 g of triethylamine. The mixture was stirred atambient temperature for 16 h. The solution was concentrated to ˜10 mLand pH was adjusted to 4-5 with acetic acid. The mixture was loaded ontoa C18 packed column, 10 g resin and eluted with water. The column waseluted with a gradient mixture of aqueous TEAA buffer and acetonitrile.The volume was 20 mL each of the following TEAA/MeCN ratio (v/v): 95/5,90/10, 85/15, 80/20, and 75/25. The appropriate fractions were combinedand concentrated to dryness, providing the product as the triethylaminesalt, 256 mg, 80%, LC/MS m/z=336.5 Da, M+1.

The solid was dissolved in 10 mL of water and potassium bicarbonate (118mg, 1.18 mmol) was added, followed by 18-crown-6 (312 mg, 1.18 mmol).The solution was concentrated to dryness. The solid was sonicated in 5mL of MTBE for 15 min. A fine slurry was obtained. The supernatant wasremoved after centrifugation. The solid was dried under vacuum toconstant weight, 550 mg.

Grignard reaction. To the suspension of the crown ether salt (190 mg,0.20 mmol) in dry THE (3 mL) was added CH₃MgCl (3 M in THF, 100 μL, 0.30mmol) and the resulting mixture was stirred at ambient temperature for 4h. To the mixture was added HCl in ether (2 M, 200 μL) and ether (3 mL).The solid was washed with 2×5 mL of dry ether and dried under vacuum.The obtained weight: 230 mg, 29% based on 0.20 mmol of startingmaterial. Product was used without further purification.

Synthesis of Quaternary Salt QS5

To the solution of benz[c,d] indol-2(1H)one (540 mg, 3.2 mmol) andmethyl bromoacetate (684 mg, 4.4 mmol) in dry DMF (10 mL) was addedpotassium carbonate (662 mg, 4.8 mmol). The mixture was stirred atambient temperature for 16 h. The reaction was quenched by addition of0.5 mL of acetic acid. The mixture was concentrated to ˜4 mL andpartitioned with ethyl acetate (30 mL) and water (20 mL). The ethylacetate solution was washed with 2×10 mL of water and 10 mL of brine.After drying over anhydrous sodium sulfate, the solution wasconcentrated to dryness. Obtained weight: 720 mg, 93%. The structure wasconfirmed by LC/MS, m/z=242.3 Da, M+1.

Sulfonation/Crown ether Salt. To starting material (483 mg, 2.0 mmol)was added chlorosulfonic acid (1.0 mL) and the mixture was stirred atambient temperature for 16 h. The solution was carefully diluted with 2mL of water and then quenched into a mixture of water/acetonitrile (2:1,v/v, 50 mL) containing 3.0 g of potassium carbonate. The mixture wasstirred at ambient temperature for 16 h. The solution was concentratedto ˜10 mL and pH was adjusted to 4-5 with acetic acid. The mixture wasloaded onto a C18 packed column, 50 g resin and eluted with 20 mL ofwater, a gradient mixture of aqueous TEAA buffer and acetonitrile. Thevolume was 20 mL each of the following TEAA/MeCN ratio (v/v): 95/5,90/10, 85/15, 80/20, and 75/25. The appropriate fractions were combinedand concentrated to dryness, providing the product as the triethylaminesalt, 557 mg, 68%, LC/MS, m/z=308.4 Da, M+1 The solid (557 mg, 1.36 mmolas triethylamine salt) was dissolved in 10 mL of water and potassiumbicarbonate (272 mg, 2.72 mmol) was added, followed by 18-crown-6 (718mg, 2.72 mmol). The solution was concentrated to dryness. The solid wassonicated in 5 mL of MTBE for 15 min. A fine slurry was obtained. Thesupernatant was removed after centrifugation. The solid was dried undervacuum to constant weight, 1.21 g.

Grignard reaction. To the suspension of the crown ether salt (365 mg,0.40 mmol) in dry THE (5 mL) was added CH₃MgCl (3 M in THF, 200 μL, 0.60mmol) and the resulting mixture was stirred at ambient temperature for16 h. To the mixture was added HCl in ether (2 M, 0.6 mL) and ether (3mL). The solid was washed with 2×5 mL of dry ether and dried undervacuum.

Weight: 490 mg, 25% based on 0.40 mmol of starting material. Product wasused without further purification.

Example 27: Dye Synthesis General Procedures

The dyes, Table 14, were synthesized by condensation of the quaternarysalt (Table 12) with the bridge (Table 13) in a mixture of aceticanhydride/acetic acid using cesium acetate as the base.

TABLE 13 Bridge Intermediates B Intermediate B Structure B₁

B₂

B₃

Symmetric Dyes

To the mixture of the quaternary salt QS (48 μmol), bridge B (20 μmol),cesium acetate (480 μmol) was added 5 mL of a mixture of aceticanhydride/acetic acid (2:1, v/v). The mixture was heated at 40-50° C.for 4-8 h. After concentrating under vacuum to ˜1 mL, water (˜10 mL) wasadded and the mixture was purified using a packed C18 short column (˜12mL bed volume), eluted with a gradient mixture of aqueous TEAA bufferand acetonitrile The volume was 20 mL each of the following TEAA/MeCNratio (v/v): 95/5, 90/10, 85/15, 80/20, and 75/25.

D81 was synthesized by condensation of QS1 with B₃, LC/MS m/z=685.2 Daand 687.2 Da, M+1, UV-Vis λ_(max)˜1026 nm (methanol).

D82 was synthesized by condensation of QS1 with B₂, LC/MS m/z=659.2 Daand 671.2 Da M+1, UV-Vis λ_(max)˜1020 nm (methanol).

D72 was synthesized by condensation of QS2 with B₃, LC/MS m/z=741.3 Daand 743 Da.

UV-Vis λ_(max)˜1030 nm (methanol).

D84 was synthesized by condensation of QS2 with B₂, LC/MS m/z=715.3 Daand 717.3 Da, M+1, UV-Vis λ_(max)˜1020 nm (methanol).

D85 was synthesized by condensation of QS3 with B₂, LC/MS m/z=875.4 Daand 877.4 Da, UV-Vis λ_(max)˜1030 nm (methanol).

D17 was synthesized by condensation of QS4 with B₁, LC/MS m/z=729.3 DaM+1, UV-Vis μ_(max)˜990 nm (methanol).

D19 was synthesized by condensation of QS5 with B₁, LC/MS m/z=673.3 Da,M+1, UV-Vis λ_(max)˜1010 nm (methanol).

D15 was synthesized by condensation of QS5 with B₃, LC/MS m/z=901.3 Da,903.1 Da, M+1, UV-Vis λ_(max)˜1030 nm (methanol).

Asymmetric Dyes

To the mixture of two different quaternary salts QS and QS' (both ˜24μmol) and the bridge B (20 μmol), cesium acetate (480 μmol) was added 5mL of a mixture of acetic anhydride/acetic acid (2:1, v/v). The mixturewas heated at 40-50° C. for 4-8 h. After concentrated under vacuum to ˜1mL, water (˜10 mL) was added and the mixture was purified usingpreparative HPLC with a C18 column (Gradient: 5-50% A/B; A=aqueous TEAAbuffer, triethylamine (12.5 mM), acetic acid (25 mM), acetonitrile 2.5%;B=acetonitrile) D13 was synthesized by condensation of QS3 and QS4 withB₁. LC/MS m/z=765.3 Da, M+1, UV-Vis λ_(max)˜980 nm (methanol).

D86 was synthesized by reaction of QS4 (24 μmol) with B₁ (20 μmol),cesium acetate (480 μmol) in 5 mL of acetic anhydride/acetic acid (2:1,v/v) at ambient temperature for 2 h. To the mixture was added QS6 (22μmol) and the reaction continued at 40° C. for 2 h. LC/MS m/z=887.3 Da,M+1, UV-Vis λ_(max)˜890 nm (methanol).

D20 was synthesized by condensation of QS1 and QS4 with B₁.LC/MSm/z=657.3 Da, M+1, UV-Vis λ_(max)˜980 nm (methanol).

D18 was synthesized by condensation of QS3 and QS5 with B₁. LC/MSm/z=737.3 Da, M+1, UV-Vis λ_(max)˜990 nm (methanol).

TABLE 14 Exemplary dyes Dye Structure D81

D82

D72

D84

D85

D17

D19

D15

D13

D86

D20

D18

Example 28: Absorbance Spectra of Fluorochrome Compounds in Methanol andWater

Equimolar concentrations of the fluorochrome compounds D3, D19 and D2were dissolved in methanol or water in a 1 mL cuvette and the absorbancemeasured against a methanol or water blank sample on a Cary 50absorbance spectrophotometer. Normalized spectra thus obtained are shownin FIG. 1 for each fluorochrome compound.

Example 29: Emission Spectra of Representative Water SolubleFluorochrome D2 in Various Solvents

The emission of D2 was measured in acetonitrile (ACN), ethanol (EtOH),methanol (MeOH). Spectra were recorded on a Quantamaster 8075-21fluorescence spectrophotometer equipped with a liquid nitrogen cooledcooled DSS-IGA020L/CUS InGaAs NIR detector with excitation at 980 nm andemission recorded from 1000 nm to 1200 nm. The emission spectra asillustrated in FIG. 2 are at equal absorbance at 980 nm for eachsolvent.

Example 30: Imaging Fluorescence Emission of D2

A 100 μL solution of fluorochrome compound D2 in water (approximately 4μM concentration) was placed in a FMT phantom and excited with a 976 nmlaser. The fluorescent light was then imaged with an InGaAs camera usinga 980 nm long pass filter.

Example 31: Fluorescence of DiSulfonated Chloro Dyes Based onbenz[cd]indoles and Comparison with IR-26 in Dichloroethane

The fluorescence spectra for an equal absorbance ˜0.13 solution of dyesA, B, C in dichloroethane were determined by exciting at 980 nm (Horiba(Quantamaster 8075-21), nitrogen cooled InGaAs detector) and comparedthem with a commercially available dye, IR-26 (obtained from Exciton) Das reference. Dye B is more than 2 fold brighter than the referenceIR-26 in DCE as illustrated in FIG. 3 .

The fluorescence of dyes A, B and C were also determined in othersolvents viz., methanol, ethanol, DMSO and water. Results areillustrated in Table 15.

TABLE 15 Fluorescence Max (nm) Solvents A B C D MeOH 1046 1056 1042 EtOH1050 1062 1046 DMSO 1080 1092 1074 DCE 1064 1080 1062 1118 Water %Fluorescence Relative to C Solvents A B C D (IR-26) MeOH 86.00 94.801.00 EtOH 92.92 76.31 1.00 DMSO 29.46 80.53 1.00 DCE 90.11 130.82 1.0058.89 Water

Dyes A, B and C have their emission highly quenched in water. Verylittle residual fluorescence is observed for an aqueous solution. Theirabsorption spectra are consistent with the blue shifted absorbance maxresulting from high degree of aggregation. Results are illustrated inFIG. 4 . From the abs and emission spectra of the disulfonatedbenz[c,d]-indole dyes, it is clear that the presence of two sulfonategroups on the fluorophores are not sufficient to have good fluorescence,which still undergo strong aggregation in aqueous solutions and resultin quenching, and require 4 or more such water solubilizing groups.

Example 32: Conjugation of Fluorophore D2 with Cyclo-(RGDyK) Peptide

The fluorophores as described herein are capable of being conjugated toan organic small molecule or a biomolecule, and the optical propertiesof the resulting conjugate are unaltered and thus may be useful forbiological applications including in vivo imaging.

An RGD peptide, cyclo-(RGDyK) was obtained from AnaSpec (Cat#AS-6113-5). It is one the most widely used imaging agents that hasheight affinity to αvβ3-integrins which are highly expressed in tumors.About one mg of compound D2 was first activated using 2 mgdisuccinikidyl carbonate (DSC) in 100 μL DMF along with 1 μL N-methylmorpholine (NMN), and incubated at 37° C. for 1 hr yielding NHSE. TheDMF solution was diluted with dry ethyl acetate (EA, 1.8 mL) and theprecipitate was centrifuged. The pellet was washed with 250 μL EA, andthe residue was speed vac dried for 20 min. The resulting NHSE wasreconstituted in 100 μL DMF and is stored at −20° C., which is stablefor weeks. 0.1 mg of D2-NHSE was reacted with 3 fold excess of RGDpeptide in 100 μL DMF and 1 μL NMM, at 37° C. After 90 min, the LCMSanalysis showed >99% conversion of the dye to the product. It waspurified on Phenylhexyl (X-Bridge) column over a gradient of 00-35% B,using 100% acetonitrile as buffer B, and 25 mM triethylammonium acetateas buffer A (pH ˜6.5).

The resulting compound was analyzed and further characterized. Purity ofD2-cyclo-(RGDyK) after HPLC purification (detection at 800 nm)illustrated no observable contamination. The calculated mass is 1610 Da(C₆₇H₇₆N₁₂O₂₅S₅) and observed mass is 806.3 Da (as half mass (M+2/2)).D2-cyclo-(RGDyK) conjugate in 1×PBS has an absorbance max at 1011 nm,and shows no “blue shift” of the absorbance peak due to aggregation inaqueous solution that is observed for the fluorophores with lesssolubilizing groups such as sulfonates. (FIG. 5 ) The fluorescenespectrum of the D2-cyclo-(RGDyK) conjugate determined on Horiba with N2cooled InGaAs detector is illustrated in FIG. 6 .

Example 33: Activation of Fluorochrome D2 to Succinimidyl Ester D80

D2 (5.5 mg) was dissolved in 100 μL of anhydrous DMF in a 2.0 mLpolypropylene centrifuge tube. DSC (disuccinimidyl carbonate, 7 mg) wasadded along with DMAP (4-dimethylaminopyridine, 0.4 mg) and NMM(N-methylmorpholine, 3 μL) and the solution was rotated at roomtemperature in the dark overnight. The material was then precipitated byaddition of 1.5 mL of ethyl acetate and centrifuged at 10,000×g for 5minutes. The ethyl acetate was decanted and the solid dispersed in 1.5mL additional ethyl acetate followed by centrifugation at 10,000 g for 5minutes and again decanting the ethyl acetate from the precipitate. Thesolid was dissolved in 100 μL of anhydrous DMF and 20 μL aliquoted intoeach of 5 centrifuge tubes containing 1.5 mL of ethyl acetate. The 5tubes were centrifuged at 10,000×g for 5 minutes then the ethyl acetatedecanted off. The precipitated solid in the tubes was dried under vacuumand the tubes containing activated N-hydroxysuccinimidyl ester D80stored at −20° C. prior to use.

Example 34: Conjugation of Succinimidyl Ester D80 to Polyethylene GlycolAmine

Methoxypolyethylene glycol amine, 40 kDa molecular weight (mPEG-NH₂-40k), was obtained from JenKemUSA (P/N M-NH2-40 k) and dissolved in 1×PBSto a concentration of 5 mg/mL. 60 μL of the mPEG-NH2-40 k solution wasplaced in a vial with 30 μL of 1×PBS. N-hydroxysuccinimidyl ester D80was dissolved in DMF to a final concentration of 10 mM and the 2.5 μLwas added to the mPEG-NH2-40 k solution followed by 10 μL of 0.1 Msodium bicarbonate, pH 8.3. The mixture was allowed to react at 23° C.for 2 hours with rotation. The labeled mPEG-NH₂-40 k was purified fromunconjugated dye and buffer components on 0.5 mL Zeba columns with 7 kDaMWCO (Thermo Fisher) according to the manufacturer's protocol afterequilibrating the columns with four 300 μL portions of 1×PBS. Afterloading the crude samples on the Zeba columns, centrifugation at 1500×gfor 120 seconds eluted the labeled mPEG-NH₂-40 k while the unconjugateddye was retained on the column.

Example 35: Optical Properties of D80 Conjugated to Polyethylene Glycol

The absorbance of purified conjugate of D80 with methoxypolyethyleneglycol amine, 40 kDa, was measured in 1×PBS solution on a Cary 50 UV/visabsorbance spectrophotometer from 250 nm to 1100 nm. Results areillustrated in FIG. 7 . The emission of the conjugate was measured in1×PBS solution on a Quantamaster 8075-21 fluorescence spectrophotometerequipped with a liquid nitrogen cooled cooled DSS-IGA020L/CUS InGaAs NIRdetector with excitation at 980 nm and emission recorded from 1000 nm to1400 nm. Results are illustrated in FIG. 8 .

Example 36: Conjugation of Succinimidyl Ester D80 to an Antibody (MouseAnti-Human IgG)

Mouse anti-human IgG was obtained from Jackson ImmunoResearch in 0.01 Mphosphate, 0.25 M NaCl, pH 7.6 at 1.7 mg/mL. 100 μL of the mouseanti-human IgG solution was placed in each of 4 vials. The conjugationof D80 to an exemplary antibody is schematically illustrated in FIG. 9 .Briefly, N-hydroxysuccinimidyl ester D80 was dissolved in DMF to a finalconcentration of 10 mM and the solution was added to the each of thefour vials (1 μL, 2 μL, 4 μL and 8 μL, respectively). The solutions wereallowed to react at 23° C. for 2 hours with rotation. The labeledantibodies were purified from unconjugated dye and buffer components on0.5 mL Zeba columns with 40 kDa MWCO (Thermo Fisher) according to themanufactures protocol after equilibrating the columns with four 300 μLportions of 1×PBS. After loading the crude samples on the Zeba columns,centrifugation at 1500×g for 120 seconds eluted the labeled antibodywhile the unconjugated dye was retained on the column. Dye loading (dyesper antibody) were calculated from absorbance at 280 nm (antibody anddye) and 1030 nm (dye) by subtracting 55% of the absorbance of the dyeat 1030 nm from the total absorbance of the sample at 280 nm (to removethe contribution of the dye to the absorbance at 280 nm) and taking theratio of the absorbance at 1030 nm to the corrected absorbance at 280nm. It was assumed that the extinction coefficients of the antibody at280 nm and the dye at 1030 nm are approximately equivalent, or about200,000 M⁻¹cm⁻¹. Dye loadings calculated in this manner for each of thefour samples were found to be 0.4, 0.6, 0.8 and 1.0, dyes per antibody,respectively.

Example 37: Optical Properties of D80 Conjugated to Mouse Anti-Human IgG

Purified mouse anti-human IgG conjugated to D2 from example 36 in 1×PBSsolution was placed in a 2 mm path length cuvette (Eppendorf UVette) andthe absorbance measured on a Cary 50 UV/vis absorbance spectrophotometerfrom 250 nm to 1100 nm. Dye loading (dyes per antibody) were calculatedfrom absorbance at 280 nm (antibody and dye) and 1030 nm (dye) bysubtracting 55% of the absorbance of the dye at 1030 nm from the totalabsorbance of the sample at 280 nm (to remove the contribution of thedye to the absorbance at 280 nm) and taking the ratio of the absorbanceat 1030 nm to the corrected absorbance at 280 nm. It was assumed thatthe extinction coefficients of the antibody at 280 nm and the dye at1030 nm are approximately equivalent, or about 200,000 M⁻¹ cm⁻¹. Dyeloadings calculated in this manner for each of the four samples werefound to be 0.4, 0.6, 0.8 and 1.0, D2 dyes per antibody, respectively.

Emission spectra of the D80 conjugated mouse anti-human IgG antibodysolutions in 1×PBS were recorded on a Quantamaster 8075-21 fluorescencespectrophotometer equipped with a liquid nitrogen cooled cooledDSS-IGA020L/CUS InGaAs NIR detector with excitation at 980 nm. Therepresentative emission spectrum of the labeled antibody (2 μL dyesolution) is illustrated in FIG. 10 .

Example 38: Conjugation of Succinimidyl Ester D80 to an Antibody(Atezolizumab)

Atezolizumab was obtained from Selleck Chemicals in 1×PBS at aconcentration of 5 mg/mL. 60 μL of the Atezolizumab antibody solution(0.3 mg, 2.1 nmol) placed in each of 3 vials, along with 30 μL of 1×PBS.N-hydroxysuccinimidyl ester D80 was dissolved in DMF to a finalconcentration of 10 mM and the solution was added to the each of thethree vials (1.25 μL, 2.5 μL and 5 μL, respectively). A solution of 0.1M sodium bicarbonate, pH 8.3 (10 μL) was then added to each of the vialsto raise the pH and the solutions were rotated at 23° C. for 2 hours.The labeled antibodies were purified from unconjugated dye and buffercomponents on 0.5 mL Zeba columns with 40 kDa MWCO (Thermo Fisher)according to the manufacturer's protocol after equilibrating the columnswith four 300 μL portions of 1×PBS. After loading the crude samples onthe Zeba columns, centrifugation at 1500×g for 120 seconds eluted thelabeled antibody while the unconjugated dye was retained on the column.The eluted sample was obtained in 1×PBS solution and filtered through a0.2 micron centrifugal filter.

Example 39: Optical Properties of D80 Conjugated to an Atezolizumab

Purified Atezolizumab conjugated to D80 from example 38 in 1×PBSsolution was placed in a 2 mm path length cuvette (Eppendorf UVette) andthe absorbance measured on a Cary 50 UV/vis absorbance spectrophotometerfrom 250 nm to 1100 nm. Dye loading (dyes per antibody) were calculatedfrom absorbance at 280 nm (antibody and dye) and 1030 nm (dye) bysubtracting 55% of the absorbance of the dye at 1030 nm from the totalabsorbance of the sample at 280 nm (to remove the contribution of thedye to the absorbance at 280 nm) and taking the ratio of the absorbanceat 1030 nm to the corrected absorbance at 280 nm. It was assumed thatthe extinction coefficients of the antibody at 280 nm and the dye at1030 nm are approximately equivalent, or about 200,000 M⁻¹cm⁻¹. Dyeloading calculated in this manner for a representative sample was foundto be approximately 0.7 dyes per antibody. The resulting spectrum isillustrated in FIG. 11 .

The emission spectrum of the D80 conjugated atezolizumab solution wasrecorded in 1×PBS solution on a Quantamaster 8075-21 fluorescencespectrophotometer equipped with a liquid nitrogen cooled cooledDSS-IGA020L/CUS InGaAs NIR detector with excitation at 980 nm. Therepresentative emission spectrum of the labeled antibody is illustratedin FIG. 12 .

Example 40: Imaging of D2 in a Mouse

Mice were injected retro-orbitally with 2 nmol of D2 in 1×PBS. The micewere subsequently sacrificed to stop blood circulation and imaged in theheart region using an InGaAs short-wave infrared camera with 976 nmlaser excitation and a 980 nm long pass filter. Fluorescent signal fromD2 was observed in the heart and carotid arteries.

Various modifications of the present invention, in addition to thoseshown and described herein, will be apparent to those skilled in the artof the above description. Such modifications are also intended to fallwithin the scope of the appended claims.

It is appreciated that all reagents are obtainable by sources known inthe art unless otherwise specified. Methods of nucleotide amplification,cell transfection, and protein expression and purification are similarlywithin the level of skill in the art.

Patents, publications, and applications mentioned in the specificationare indicative of the levels of those skilled in the art to which theinvention pertains. These patents, publications, and applications areincorporated herein by reference to the same extent as if eachindividual patent, publication, or application was specifically andindividually incorporated herein by reference.

The foregoing description is illustrative of particular embodiments ofthe invention, but is not meant to be a limitation upon the practicethereof. The following claims, including all equivalents thereof, areintended to define the scope of the invention.

1. A compound represented by one of the following structures:

or a salt thereof, wherein: R₁ is independently for each occurrence,hydrogen substituted or unsubstituted C₁ to C₂₄ alkyl, substituted orunsubstituted alkylaryl, substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl, substituted or unsubstituted alkenyl,substituted or unsubstituted alkynyl, sulfonate, aryl sulfonate, alkylsulfonate, taurine, carboxylate, amine, alkylamine, arylamine,alkylammonium, arylammonium, sulfonamide, halogen, hydroxy, amide,nitro, cyano, azide, O-alkyl, S-alkyl, silyl, trialkylsilyl, O-silyl,haloalkyl, alkylsulfhydryl, trifluoromethyl, hydrazide, substituted orunsubstituted aryl, heteroaryl, or heterocyclic (e.g. morpholine)alkynyl, carboxyalkyl, aminoalkyl, haloalkyl, azidoalkyl, amide, aminoacid, or peptide, O, S, N, P, Si, C, (C═C), or L; L is absent or is alinker moiety, optionally bearing a functional group or reactive group,wherein said functional group or reactive group is a carboxylate,carboxyalkyl, maleimide, succinimidyl ester, carboxamide, propargyl,azidoalkyl, alkyne, isothiocyanate, of —NH₂—OH, —SH, —SO₃H, carboxyl,—COCl, —CONHNH₂, acetoxymethyl esters, substituted and unsubstitutedN-hydroxysuccinimidyl esters, substituted and unsubstitutedN-hydroxysulfosuccinimido esters, nitro- or fluoro or phenol esters,azide, —COCH₂I, phosphoramidite, phthalamido, acyl fluoride, acylchloride, acyl azide, tyramide, cinnamamide, hydroxycinnamamide,aldehyde, ketone, phosphoramidite, isocyanate, isothiocyanate, sulfonylchloride, maleimide or biotin; R₂ is a substituted or unsubstituted C₁to C₂₄ alkyl, substituted or unsubstituted alkylaryl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylsulfonate, C₁ to C₂₄ alkyl sulfonate, C₁-C₂₄ alkyl carboxylate, arylcarboxylate, C₁-C₂₄ alkylamine, arylamine, C₁-C₂₄ alkylammonium,arylammonium, or substituted or unsubstituted polyethylene glycol. 2.The compound of claim 1, wherein R₂ is a substituted or unsubstituted C₁to C₂₄ alkyl, substituted or unsubstituted alkylaryl, substituted orunsubstituted aryl, aryl sulfonate, C₁ to C₂₄ alkyl sulfonate, C₁-C₂₄alkyl carboxylate, or aryl carboxylate.
 3. The compound of claim 1,wherein R₁ is in each occurrence a sulfonate, alkyl sulfonate,arylsulfonate or taurine.
 4. The compound of claim 1, wherein: R₁ is ineach occurrence a sulfonate, alkyl sulfonate, arylsulfonate or taurine;and R₂ is a substituted or unsubstituted C₁ to C₂₄ alkyl, substituted orunsubstituted alkylaryl, substituted or unsubstituted aryl, arylsulfonate, C₁ to C₂₄ alkyl sulfonate, C₁-C₂₄ alkyl carboxylate, or arylcarboxylate.
 5. The compound of claim 4, wherein R₁ in each occurrenceis a sulfonate.